Bckdk inhibitors

ABSTRACT

wherein R1, R2, and R3 are defined herein, their use as branched-chain alpha keto acid dehydrogenase kinase inhibitors, pharmaceutical compositions containing such inhibitors and the use of such inhibitors to treat, for example, diabetes, NASH and heart failure.

FIELD OF THE INVENTION

The present invention provides compounds that are branched-chain alphaketo acid dehydrogenase kinase inhibitors, pharmaceutical compositionscontaining such inhibitors and the use of such inhibitors to treat forexample, diabetes, NASH and heart failure.

BACKGROUND OF THE INVENTION

Branched-chain amino acids (BCAAs) account for about 40% of theessential amino acids in healthy subjects and must be acquired through awell-balanced diet. Branched-chain amino acids are toxic in excess butare required for protein synthesis and cellular signaling processes.BCAAs are transaminated by branched-chain aminotransferase (BOAT) totheir alpha-keto acid forms: alpha-ketoisocaproate (KIC/ketoleucine),2-keto-3-methylvalerate (KMV/ketoisoleucine) and alpha-ketoisovalerate(KIV/ketovaline). The branched-chain keto acids (BCKAs) are thenoxidatively decarboxylated by the branched-chain ketoacid dehydrogenase(BCKDH) enzyme complex, which consists of multiple copies of BCKDH E1α/βtetramers, BCKDH E2, and BCKDH E3 subunits. The complex is regulated byinhibitory phosphorylation, which is mediated by BCKDH kinase (BCKDK),and this same phosphorylation site is dephosphorylated by thephosphatase PPM1K. Inhibition of complex phosphorylation promotes BCKDHactivity and thus the irreversible catabolism of BCKA. (Lynch C J, AdamsS H: Branched-chain amino acids in metabolic signalling and insulinresistance. Nat Rev Endocrinol 2014, 10:723-36.) Deletion of Bckdk inmice confirms this regulation as mice lacking Bckdk display increasedBCKDH activity in multiple tissues. (Joshi M A, Jeoung N H, Obayashi M,Hattab E M, Brocken E G, Liechty E A, Kubek M J, Vattem K M, Wek R C,Harris R A: Impaired growth and neurological abnormalities inbranched-chain alpha-keto acid dehydrogenase kinase-deficient mice.Biochem J 2006, 400:153-62.)

U.S. Pat. No. 9,078,865 is directed to for example, methods ofdecreasing plasma levels of one or more branched-chain amino acids orbranched-chain alpha-ketoacids comprising administering to an individualin need thereof a therapeutically effective amount of at least onecompound of the formula: phenyl-CH₂—(CH₂)_(n)—COOH wherein n is 0, 2, 4,6 or 8 in order to treat for example an inborn error of metabolism innewborns known as maple syrup urine disease (MSUD). MSUD, also calledbranched-chain ketoaciduria, is an autosomal recessive disorder.

There is a strong correlation with BCAA catabolism and cardiometabolichealth. Increased BCAA/BCKA levels have been observed in plasma of type2 diabetic patients in multiple studies. (Wang T J, Larson M G, Vasan RS, Cheng S, Rhee E P, McCabe E, Lewis G D, Fox C S, Jacques P F,Fernandez C, O'Donnell C J, Carr S A, Mootha V K, Florez J C, Souza A,Melander O, Clish C B, Gerszten R E: Metabolite profiles and the risk ofdeveloping diabetes. Nat Med 2011, 17:448-53; Newgard C B, An J, Bain JR, Muehlbauer M J, Stevens R D, Lien L F, Haqq A M, Shah S H, Arlotto M,Slentz C A, Rochon J, Gallup D, Ilkayeva O, Wenner B R, Yancy W S, Jr.,Eisenson H, Musante G, Surwit R S, Millington D S, Butler M D, Svetkey LP: A branched-chain amino acid-related metabolic signature thatdifferentiates obese and lean humans and contributes to insulinresistance. Cell Metab 2009, 9:311-26.)

Reduced PPM1K and increased BCKDK levels were observed in human NASH.(Lake A D, Novak P, Shipkova P, Aranibar N, Robertson D G, Reily M D,Lehman-McKeeman L D, Vaillancourt R R, Cherrington N J: Branched chainamino acid metabolism profiles in progressive human nonalcoholic fattyliver disease. Amino Acids 2015, 47:603-15.)

Reduced mRNA levels for enzymes in the catabolic pathway have also beenobserved in skeletal muscle of human diabetic patients. (Lerin C,Goldfine A B, Boes T, Liu M, Kasif S, Dreyfuss J M, De Sousa-Coelho A L,Daher G, Manoli I, Sysol J R, Isganaitis E, Jessen N, Goodyear L J,Beebe K, Gall W, Venditti C P, Patti M E: Defects in musclebranched-chain amino acid oxidation contribute to impaired lipidmetabolism. Mol Metab 2016, 5:926-36.)

Similarly, metabolomics and RNA profiling data from mouse hearts alsosuggest that genes in the BCAA/BCKA catabolic pathway are downregulatedin heart failure. (Lai L, Leone T C, Keller M P, Martin O J, Broman A T,Nigro J, Kapoor K, Koves T R, Stevens R, Ilkayeva O R, Vega R B, Attie AD, Muoio D M, Kelly D P: Energy metabolic reprogramming in thehypertrophied and early stage failing heart: a multisystems approach.Circ Heart Fail 2014, 7:1022-31; Sun H, Olson K C, Gao C, Prosdocimo DA, Zhou M, Wang Z, Jeyaraj D, Youn J Y, Ren S, Liu Y, Rau C D, Shah S,Ilkayeva O, Gui W J, William N S, Wynn R M, Newgard C B, Cai H, Xiao X,Chuang D T, Schulze P C, Lynch C, Jain M K, Wang Y: Catabolic Defect ofBranched-Chain Amino Acids Promotes Heart Failure. Circulation 2016,133:2038-49.)

These data collectively suggest that BCAA catabolism is impaired inmultiple human disease states. One mechanism to increase BCAA catabolismis a BCKDK inhibitor. By inhibiting BCKDK, BCKDH activity will increaseand BCAA catabolism will be increased. Another mechanism to increaseBCAA catabolism is a BCKDK degrader. By degrading BCKDK, BCKDH activitywill increase and BCAA catabolism will be increased. Although there hasbeen some early research related to BCKDK there remains a need forpharmaceutical agents that have BCKDK inhibiting and/or degradingactivity and are useful in the treatment, prevention or diminution ofthe manifestations of the maladies described herein.

SUMMARY OF THE INVENTION

The present invention is directed to compounds of Formula I

wherein

R¹ is H, bromo, chloro, fluoro, (C₁-C₂)alkyl, or (C₁-C₂)fluoroalkyl;

R² is fluoro or chloro, wherein if R¹ is chloro and R³ is H then R² isfluoro; and

R³ is H, chloro, fluoro, methyl, or (C₁)fluoroalkyl, wherein if R¹ is Hthen R³ is chloro, fluoro, methyl or (C₁)fluoroalkyl;

or a pharmaceutically acceptable salt of said compound.

The present invention is also directed at methods of treating fattyliver, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis,non-alcoholic steatohepatitis with liver fibrosis, nonalcoholicsteatohepotitis with cirrhosis or nonalcoholic steatohepatitis withcirrhosis and hepatocellular carcinoma including administering to amammal, such as a human, in need of such treatment a therapeuticallyeffective amount of a compound of Formula I or a pharmaceuticallyacceptable salt of said compound.

The present invention is also directed at methods of treating heartfailure, congestive heart failure, coronary heart disease, peripheralvascular disease, renovascular disease, pulmonary hypertension,vasculitis, acute coronary syndromes and modification of cardiovascularrisk including administering to a mammal, such as a human, in need ofsuch treatment a therapeutically effective amount of a compound ofFormula I or a pharmaceutically acceptable salt of said compound.

The present invention is also directed at methods of treating Type Idiabetes, Type II diabetes mellitus, idiopathic Type I diabetes (TypeIb), latent autoimmune diabetes in adults (LADA), early-onset Type 2diabetes (EOD), youth-onset atypical diabetes (YOAD), maturity onsetdiabetes of the young (MODY), malnutrition-related diabetes, gestationaldiabetes, coronary heart disease, ischemic stroke, restenosis afterangioplasty, peripheral vascular disease, intermittent claudication,myocardial infarction, dyslipidemia, post-prandial lipemia, conditionsof impaired glucose tolerance (IGT), conditions of impaired fastingplasma glucose, metabolic acidosis, ketosis, arthritis, diabeticretinopathy, macular degeneration, cataract, diabetic nephropathy,glomerulosclerosis, chronic renal failure, diabetic neuropathy,metabolic syndrome, syndrome X, hyperglycemia, hyperinsulinemia,hypertriglyceridemia, insulin resistance, impaired glucose metabolism,skin and connective tissue disorders, foot ulcerations and ulcerativecolitis, endothelial dysfunction and impaired vascular compliance, hyperapo B lipoproteinemia, and maple syrup urine disease includingadministering to a mammal, such as a human, in need of such treatment atherapeutically effective amount of a compound of Formula I or apharmaceutically acceptable salt of said compound.

The present invention is also directed at methods of treatinghepatocellular carcinoma, kidney renal clear cell carcinoma, head andneck squamous cell carcinoma, colorectal adenocarcinoma, mesothelioma,stomach adenocarcinoma, adrenocortical carcinoma, kidney papillary cellcarcinoma, cervical and endocervical carcinoma, bladder urothelialcarcinoma, lung adenocarcinoma including administering to a mammal, suchas a human, in need of such treatment a therapeutically effective amountof a compound of Formula I or a pharmaceutically acceptable salt of saidcompound.

The present invention is also directed at pharmaceutical compositionshaving a therapeutically effective amount of a compound of Formula I ora pharmaceutically acceptable salt of said compound and apharmaceutically acceptable carrier, vehicle or diluent.

The present invention is also directed at pharmaceutical combinationcompositions that include: a therapeutically effective amount of acomposition having:

a first compound, said first compound being a compound of Formula I or apharmaceutically acceptable salt of said compound;

a second compound, said second compound being an anti-diabetic agent; anon-alcoholic steatohepatitis treatment agent, a non-alcoholic fattyliver disease treatment agent or an anti-heart failure treatment agentand

a pharmaceutical carrier, vehicle or diluent.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a characteristic X-ray powder diffraction pattern showingExample 1, Form 1 (Vertical Axis: Intensity (CPS); Horizontal Axis: Twotheta (degrees)).

FIG. 2 is a characteristic X-ray powder diffraction pattern showingExample 2, Form 1 (Vertical Axis: Intensity (CPS); Horizontal Axis: Twotheta (degrees)).

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of exemplary embodiments of the inventionand the examples included therein.

It is to be understood that this invention is not limited to specificsynthetic methods of making that may of course vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting. In this specification and in the claims that follow, referencewill be made to a number of terms that shall be defined to have thefollowing meanings:

As used herein in the specification, “a” or “an” may mean one or more.As used herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more.

The term “about” refers to a relative term denoting an approximation ofplus or minus 10% of the nominal value it refers, in one embodiment, toplus or minus 5%, in another embodiment, to plus or minus 2%. For thefield of this disclosure, this level of approximation is appropriateunless the value is specifically stated to require a tighter range.

The term “alkyl”, alone or in combination, means an acyclic, saturatedhydrocarbon group of the formula CnH2n+1 which may be linear orbranched. Examples of such groups include methyl, ethyl, n-propyl,isopropyl, butyl, sec-butyl, isobutyl and t-butyl. The carbon atomcontent of alkyl and various other hydrocarbon-containing moieties isindicated by a prefix designating a lower and upper number of carbonatoms in the moiety, that is, the prefix Ci-Cj indicates a moiety of theinteger “i” to the integer “j” carbon atoms, inclusive. Thus, forexample, C₁-C₃ alkyl refers to alkyl of one to three carbon atoms,inclusive.

“Fluoroalkyl” means an alkyl as defined herein substituted with one, twoor three fluoro atoms. Exemplary (C₁)fluoroalkyl compounds includefluoromethyl, difluoromethyl and trifluoromethyl; exemplary(C₂)fluoroalkyl compounds include 1-fluoroethyl, 2-fluoroethyl,1,1-difluoroethyl, 1,2-difluoroethyl, 1,1,1-trifluoroethyl,1,1,2-trifluoroethyl, and the like.

“Compounds” when used herein includes any pharmaceutically acceptablederivative or variation, including conformational isomers (e.g., cis andtrans isomers) and all optical isomers (e.g., enantiomers anddiastereomers), racemic, diastereomeric and other mixtures of suchisomers, as well as solvates, hydrates, isomorphs, polymorphs,tautomers, esters, salt forms, and prodrugs. The expression “prodrug”refers to compounds that are drug precursors which followingadministration, release the drug in vivo via some chemical orphysiological process (e.g., a prodrug on being brought to thephysiological pH or through enzyme action is converted to the desireddrug form). Exemplary prodrugs upon cleavage release the correspondingfree acid, and such hydrolyzable ester-forming residues of the compoundsof the present invention include but are not limited to those having acarboxyl moiety wherein the free hydrogen is replaced by (C₁-C₄)alkyl,(C₂-C₇)alkanoyloxymethyl, 1-(alkanoyloxy)ethyl having from 4 to 9 carbonatoms, 1-methyl-1-(alkanoyloxy)-ethyl having from 5 to 10 carbon atoms,alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms,1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms,1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms,N(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms,1-(N(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms,3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl,di-N,N—(C₁-C₂)alkylamino(C₂-C₃)alkyl (such as β-dimethylaminoethyl),carbamoyl-(C₁-C₂)alkyl, N,N-di(C₁-C₂)alkylcarbamoyl-(C₁-C₂)alkyl andpiperidino-, pyrrolidino- or morpholino(C₂-C₃)alkyl.

The term “mammal” refers to human, livestock or companion animals.

The term “companion animal” or “companion animals” refers to animalskept as pets or household animal. Examples of companion animals includedogs, cats, and rodents including hamsters, guinea pigs, gerbils and thelike, rabbits, and ferrets.

The term “livestock” refers to animals reared or raised in anagricultural setting to make products such as food or fiber, or for itslabor. In some embodiments, livestock are suitable for consumption bymammals, for example humans. Examples of livestock animals includecattle, goats, horses, pigs, sheep, including lambs, and rabbits.

“Patient” refers to warm blooded animals such as, for example, guineapigs, mice, rats, gerbils, cats, rabbits, dogs, cattle, goats, sheep,horses, monkeys, chimpanzees, and humans.

The term “treating” or “treatment” means an alleviation of symptomsassociated with a disease, disorder or condition, or halt of furtherprogression or worsening of those symptoms. Depending on the disease andcondition of the patient, the term “treatment” as used herein mayinclude one or more of curative, palliative and prophylactic treatment.Treatment can also include administering a pharmaceutical formulation ofthe present invention in combination with other therapies.

“Therapeutically effective amount” means an amount of a compound of thepresent invention that (i) treats or prevents the particular disease,condition, or disorder, (ii) attenuates, ameliorates, or eliminates oneor more symptoms of the particular disease, condition, or disorder, or(iii) prevents or delays the onset of one or more symptoms of theparticular disease, condition, or disorder described herein.

The term “pharmaceutically acceptable” means the substance (e.g., thecompounds of the invention) and any salt thereof, or compositioncontaining the substance or salt of the invention that is suitable foradministration to a patient.

One embodiment of the present invention includes compounds of Formula Iwherein R¹ is H, bromo or chloro; R² is fluoro; and R³ is H or fluorowherein if R¹ is H then R³ is fluoro; or a pharmaceutically acceptablesalt of said compound.

Another embodiment of the present invention includes compounds havingthe structure

and crystals including said compounds or pharmaceutically acceptablesalts thereof.

Another embodiment of the present invention includes use of a compoundof Formula I or a pharmaceutically acceptable salt of said compound foruse as a medicament in treating fatty liver, nonalcoholic fatty liverdisease, non-alcoholic steatohepatitis, nonalcoholic steatohepatitiswith liver fibrosis, non-alcoholic steatohepotitis with cirrhosis ornonalcoholic steatohepatitis with cirrhosis and hepatocellular carcinomaincluding administering to a mammal, such as a human, in need of suchtreatment a therapeutically effective amount.

Another embodiment of the present invention includes use of a compoundof Formula I or a pharmaceutically acceptable salt of said compound forthe manufacture of a medicament in treating fatty liver, nonalcoholicfatty liver disease, non-alcoholic steatohepatitis, nonalcoholicsteatohepatitis with liver fibrosis, non-alcoholic steatohepotitis withcirrhosis or nonalcoholic steatohepatitis with cirrhosis andhepatocellular carcinoma including administering to a mammal, such as ahuman, in need of such treatment a therapeutically effective amount.

Another embodiment of the present invention includes use of a compoundof Formula I or a pharmaceutically acceptable salt of said compound foruse as a medicament in treating heart failure, congestive heart failure,coronary heart disease, peripheral vascular disease, renovasculardisease, pulmonary hypertension, vasculitis, acute coronary syndromesand modification of cardiovascular risk including administering to amammal, such as a human, in need of such treatment a therapeuticallyeffective amount of a compound of Formula I or a pharmaceuticallyacceptable salt of said compound.

Another embodiment of the present invention includes use of a compoundof Formula I or a pharmaceutically acceptable salt of said compound forthe manufacture of a medicament in treating heart failure, congestiveheart failure, coronary heart disease, peripheral vascular disease,renovascular disease, pulmonary hypertension, vasculitis, acute coronarysyndromes and modification of cardiovascular risk includingadministering to a mammal, such as a human, in need of such treatment atherapeutically effective amount of a compound of Formula I or apharmaceutically acceptable salt of said compound.

Another embodiment of the present invention includes use of a compoundof Formula I or a pharmaceutically acceptable salt of said compound foruse as a medicament in treating Type I diabetes, Type II diabetesmellitus, idiopathic Type I diabetes (Type Ib), latent autoimmunediabetes in adults (LADA), early-onset Type 2 diabetes (EOD),youth-onset atypical diabetes (YOAD), maturity onset diabetes of theyoung (MODY), malnutrition-related diabetes, gestational diabetes,coronary heart disease, ischemic stroke, restenosis after angioplasty,peripheral vascular disease, intermittent claudication, myocardialinfarction, dyslipidemia, post-prandial lipemia, conditions of impairedglucose tolerance (IGT), conditions of impaired fasting plasma glucose,metabolic acidosis, ketosis, arthritis, diabetic retinopathy, maculardegeneration, cataract, diabetic nephropathy, glomerulosclerosis,chronic renal failure, diabetic neuropathy, metabolic syndrome, syndromeX, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, insulinresistance, impaired glucose metabolism, skin and connective tissuedisorders, foot ulcerations and ulcerative colitis, endothelialdysfunction and impaired vascular compliance, hyper apo Blipoproteinemia, and maple syrup urine disease including administeringto a mammal, such as a human, in need of such treatment atherapeutically effective amount of a compound of Formula I or apharmaceutically acceptable salt of said compound.

Another embodiment of the present invention includes use of a compoundof Formula I or a pharmaceutically acceptable salt of said compound forthe manufacture of a medicament in treating Type I diabetes, Type IIdiabetes mellitus, idiopathic Type I diabetes (Type Ib), latentautoimmune diabetes in adults (LADA), early-onset Type 2 diabetes (EOD),youth-onset atypical diabetes (YOAD), maturity onset diabetes of theyoung (MODY), malnutrition-related diabetes, gestational diabetes,coronary heart disease, ischemic stroke, restenosis after angioplasty,peripheral vascular disease, intermittent claudication, myocardialinfarction, dyslipidemia, post-prandial lipemia, conditions of impairedglucose tolerance (IGT), conditions of impaired fasting plasma glucose,metabolic acidosis, ketosis, arthritis, diabetic retinopathy, maculardegeneration, cataract, diabetic nephropathy, glomerulosclerosis,chronic renal failure, diabetic neuropathy, metabolic syndrome, syndromeX, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, insulinresistance, impaired glucose metabolism, skin and connective tissuedisorders, foot ulcerations and ulcerative colitis, endothelialdysfunction and impaired vascular compliance, hyper apo Blipoproteinemia, and maple syrup urine disease including administeringto a mammal, such as a human, in need of such treatment atherapeutically effective amount of a compound of Formula I or apharmaceutically acceptable salt of said compound.

Another embodiment of the present invention includes use of a compoundof Formula I or a pharmaceutically acceptable salt of said compound foruse as a medicament in treating hepatocellular carcinoma, kidney renalclear cell carcinoma, head and neck squamous cell carcinoma, colorectaladenocarcinoma, mesothelioma, stomach adenocarcinoma, adrenocorticalcarcinoma, kidney papillary cell carcinoma, cervical and endocervicalcarcinoma, bladder urothelial carcinoma, lung adenocarcinoma includingadministering to a mammal, such as a human, in need of such treatment atherapeutically effective amount of a compound of Formula I or apharmaceutically acceptable salt of said compound.

Another embodiment of the present invention includes use of a compoundof Formula I or a pharmaceutically acceptable salt of said compound forthe manufacture of a medicament in treating hepatocellular carcinoma,kidney renal clear cell carcinoma, head and neck squamous cellcarcinoma, colorectal adenocarcinoma, mesothelioma, stomachadenocarcinoma, adrenocortical carcinoma, kidney papillary cellcarcinoma, cervical and endocervical carcinoma, bladder urothelialcarcinoma, lung adenocarcinoma including administering to a mammal, suchas a human, in need of such treatment a therapeutically effective amountof a compound of Formula I or a pharmaceutically acceptable salt of saidcompound.

The present invention includes all pharmaceutically acceptableisotopically-labelled compounds of Formula I wherein one or more atomsare replaced by atoms having the same atomic number, but an atomic massor mass number different from the atomic mass or mass number usuallyfound in nature.

Examples of isotopes suitable for inclusion in the compounds of theinvention include isotopes of hydrogen, such as ²H and ³H, carbon, suchas ¹¹C, ¹³C and ¹⁴C, chlorine, such as ³⁶Cl, fluorine, such as ¹⁸F,oxygen, such as ¹⁵O, ¹⁷O and ¹⁸O, and sulphur, such as ³⁵S.

Certain isotopically-labelled compounds of Formula I for example, thoseincorporating a radioactive isotope, are useful in drug and/or substratetissue distribution studies. The radioactive isotopes tritium, i.e. ³H,and carbon-14, i.e. ¹⁴C, are particularly useful for this purpose inview of their ease of incorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium, i.e. ²H, mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, and ¹⁵O,can be useful in Positron Emission Tomography (PET) studies forexamining substrate receptor occupancy.

Isotopically-labelled compounds of Formula I can generally be preparedby conventional techniques known to those skilled in the art or byprocesses analogous to those described in the accompanying Examples andPreparations using an appropriate isotopically-labelled reagents inplace of the non-labelled reagent previously employed.

Certain compounds of the present invention may exist in more than onecrystal form (generally referred to as “polymorphs”). Polymorphs may beprepared by crystallization under various conditions, for example, usingdifferent solvents or different solvent mixtures for recrystallization;crystallization at different temperatures; and/or various modes ofcooling, ranging from very fast to very slow cooling duringcrystallization. Polymorphs may also be obtained by heating or meltingthe compound of the present invention followed by gradual or fastcooling. The presence of polymorphs may be determined by solid probe NMRspectroscopy, IR spectroscopy, differential scanning calorimetry, powderX-ray diffraction or such other techniques.

Salts encompassed within the term “pharmaceutically acceptable salts”refer to the compounds of this invention which are generally prepared byreacting the free acid with a suitable organic or inorganic base toprovide a salt of the compound of the invention that is suitable foradministration to a patient.

Suitable base salts are formed from bases which form non-toxic salts.Examples include the aluminium, arginine, calcium, choline,diethylamine, glycine, lysine, magnesium, meglumine, olamine, potassium,sodium, trimethamine and zinc salts. Hemisalts of acids and bases mayalso be formed, for example, hemisulfate and hemicalcium salts. For areview on suitable salts, see Handbook of Pharmaceutical Salts:Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002).

Hemisalts of bases may also be formed, for example, hemicalcium salts.For a review on suitable salts, see Handbook of Pharmaceutical Salts:Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002).

Pharmaceutically acceptable salts of compounds of Formula I may beprepared by one or more of three methods:

-   (i) by reacting the compound of Formula I with the desired base;-   (ii) by removing a base-labile protecting group from a suitable    precursor of the compound of the invention or by ring-opening a    suitable cyclic precursor, for example, a lactone or lactam, using    the desired base; or-   (iii) by converting one salt of the compound of the invention to    another by reaction with an appropriate base or by means of a    suitable ion exchange column.

All three reactions are typically carried out in solution. The resultingsalt may precipitate out and be collected by filtration or may berecovered by evaporation of the solvent. The degree of ionization in theresulting salt may vary from completely ionized to almost non-ionized.

The compounds of Formula I, and pharmaceutically acceptable saltsthereof, may exist in unsolvated and solvated forms. The term ‘solvate’is used herein to describe a molecular complex comprising the compoundof Formula I, or a pharmaceutically acceptable salt thereof, and one ormore pharmaceutically acceptable solvent molecules, for example,ethanol. The term ‘hydrate’ is employed when said solvent is water.

A currently accepted classification system for organic hydrates is onethat defines isolated site, channel, or metal-ion coordinatedhydrates—see Polymorphism in Pharmaceutical Solids by K. R. Morris (Ed.H. G. Brittain, Marcel Dekker, 1995). Isolated site hydrates are ones inwhich the water molecules are isolated from direct contact with eachother by intervening organic molecules. In channel hydrates, the watermolecules lie in lattice channels where they are next to other watermolecules. In metal-ion coordinated hydrates, the water molecules arebonded to the metal ion.

When the solvent or water is tightly bound, the complex may have awell-defined stoichiometry independent of humidity. When, however, thesolvent or water is weakly bound, as in channel solvates and hygroscopiccompounds, the water/solvent content may be dependent on humidity anddrying conditions. In such cases, non-stoichiometry will be the norm.

Also included within the scope of the invention are multi-componentcomplexes (other than salts and solvates) wherein the drug and at leastone other component are present in stoichiometric or non-stoichiometricamounts. Complexes of this type include clathrates (drug-host inclusioncomplexes) and co-crystals. The latter are typically defined ascrystalline complexes of neutral molecular constituents which are boundtogether through non-covalent interactions, but could also be a complexof a neutral molecule with a salt. Co-crystals may be prepared by meltcrystallization, by recrystallization from solvents, or by physicallygrinding the components together—see Chem Commun, 17, 1889-1896, by O.Almarsson and M. J. Zaworotko (2004). For a general review ofmulti-component complexes, see J Pharm Sci, 64 (8), 1269-1288, byHaleblian (August 1975).

Also included within the scope of the invention are active metabolitesof compounds of Formula I (including prodrugs), that is, compoundsformed in vivo upon administration of the drug, often by oxidation ordealkylation. An example of metabolites in accordance with the inventionincludes where the compound of Formula I contains a methyl group, ahydroxymethyl derivative thereof (—CH₃->—CH₂OH):

The compounds of the invention may exist in a continuum of solid statesranging from fully amorphous to fully crystalline. The term ‘amorphous’refers to a state in which the material lacks long-range order at themolecular level and, depending upon temperature, may exhibit thephysical properties of a solid or a liquid. Typically such materials donot give distinctive X-ray diffraction patterns and, while exhibitingthe properties of a solid, are more formally described as a liquid. Uponheating, a change from solid to liquid properties occurs which ischaracterized by a change of state, typically second order (‘glasstransition’). The term ‘crystalline’ refers to a solid phase in whichthe material has a regular ordered internal structure at the molecularlevel and gives a distinctive X-ray diffraction pattern with definedpeaks. Such materials when heated sufficiently will also exhibit theproperties of a liquid, but the change from solid to liquid ischaracterised by a phase change, typically first order (‘meltingpoint’).

The compounds of Formula I may also exist in a mesomorphic state(mesophase or liquid crystal) when subjected to suitable conditions. Themesomorphic state is intermediate between the true crystalline state andthe true liquid state (either melt or solution). Mesomorphism arising asthe result of a change in temperature is described as ‘thermotropic’ andthat resulting from the addition of a second component, such as water oranother solvent, is described as ‘lyotropic’. Compounds that have thepotential to form lyotropic mesophases are described as ‘amphiphilic’and consist of molecules which possess an ionic (such as —COO⁻Na⁺,—COO⁻K⁺, or —SO₃Na⁺) or non-ionic (such as —N⁻N⁺(CH₃)₃) polar headgroup. For more information, see Crystals and the Polarizing Microscopeby N. H. Hartshorne and A. Stuart, 4th Edition (Edward Arnold, 1970).

The compounds of Formula I may exhibit polymorphism and/or one or morekinds of isomerism (e.g. optical, geometric or tautomeric isomerism).The compounds of Formula I may also be isotopically labelled. Suchvariation is implicit to the compounds of Formula I defined as they areby reference to their structural features and therefore within the scopeof the invention.

The term “room temperature or ambient temperature” means a temperaturebetween 18 to 25° C., “HPLC” refers to high-pressure liquidchromatography, “MPLC” refers to medium-pressure liquid chromatography,“TLC” refers to thin-layer chromatography, “MS” refers to mass spectrumor mass spectroscopy or mass spectrometry, “NMR” refers to nuclearmagnetic resonance spectroscopy, “DCM” refers to dichloromethane, “DMSO”refers to dimethyl sulfoxide, “DME” refers to 1,2-dimethoxyethane,“EtOAc” refers to ethyl acetate, “MeOH” refers to methanol, “Ph” refersto the phenyl group, “Pr” refers to propyl, “trityl” refers to thetriphenylmethyl group, “ACN” refers to acetonitrile, “DEAD” refers todiethyl azodicarboxylate, and “DIAD” refers to diisopropylazodicarboxylate.

In general the compounds of this invention can be made by processeswhich include processes analogous to those known in the chemical arts,particularly in light of the description contained herein. Certainprocesses for the manufacture of the compounds of this invention areprovided as further features of the invention and are illustrated by thefollowing reaction schemes. Other processes may be described in theexperimental section. Specific synthetic schemes for preparation of thecompounds of Formula I are outlined below.

As used herein, the expressions “reaction-inert solvent” and “inertsolvent” refer to a solvent or a mixture thereof which does not interactwith starting materials, reagents, intermediates or products in a mannerwhich adversely affects the yield of the desired product.

As an initial note, in the preparation of the Formula I compounds it isnoted that some of the preparation methods useful for the preparation ofthe compounds described herein may require protection of remotefunctionality (e.g., primary amine, secondary amine, carboxyl in FormulaI precursors). The need for such protection will vary depending on thenature of the remote functionality and the conditions of the preparationmethods. The need for such protection is readily determined by oneskilled in the art. The use of such protection/deprotection methods isalso within the skill in the art. For a general description ofprotecting groups and their use, see T. W. Greene, Protective Groups inOrganic Synthesis, John Wiley & Sons, New York, 1991.

For example, certain compounds contain primary amines or carboxylic acidfunctionalities which may interfere with reactions at other sites of themolecule if left unprotected. Accordingly, such functionalities may beprotected by an appropriate protecting group which may be removed in asubsequent step. Suitable protecting groups for amine and carboxylicacid protection include those protecting groups commonly used in peptidesynthesis (such as N-tert-butoxycarbonyl, benzyloxycarbonyl, and9-fluorenylmethylenoxycarbonyl for amines and lower alkyl or benzylesters for carboxylic acids), which are generally not chemicallyreactive under the reaction conditions described and can typically beremoved without chemically altering other functionality in the Formula Icompound.

Compounds of Formula I may be prepared according to the Examplesprovided herein.

The starting materials and reagents for the above described Formula Icompounds are also readily available or can be easily synthesized bythose skilled in the art using conventional methods of organicsynthesis. For example, many of the compounds used herein, are relatedto, or are derived from compounds in which there is a large scientificinterest and commercial need, and accordingly many such compounds arecommercially available or are reported in the literature or are easilyprepared from other commonly available substances by methods which arereported in the literature.

The present invention is also directed at pharmaceutical compositionshaving a therapeutically effective amount of a compound of Formula I ora pharmaceutically acceptable salt of said compound and apharmaceutically acceptable carrier, vehicle or diluent.

The compounds of this invention may also be used in conjunction withother pharmaceutical agents (e.g., antiatherosclerotic andantithrombotic agents) for the treatment of the disease/conditionsdescribed herein. The present invention is also directed atpharmaceutical combination compositions that include: a therapeuticallyeffective amount of a composition having:

a first compound, said first compound being a compound of any of FormulaI or a pharmaceutically acceptable salt of said compound;

a second compound, said second compound being an anti-diabetic agent; anon-alcoholic steatohepatitis treatment agent, a non-alcoholic fattyliver disease treatment agent or an anti-heart failure treatment agentand

a pharmaceutical carrier, vehicle or diluents.

In one embodiment of the present invention, said non-alcoholicsteatohepatitis treatment agent or non-alcoholic fatty liver diseasetreatment agent is an ACC inhibitor, a KHK inhibitor, a DGAT-2inhibitor, an FXR agonist, metformin, incretin analogs, or an incretinreceptor modulator.

In another embodiment of the present invention, said anti-diabetic agentis an SGLT-2 inhibitor, metformin, incretin analogs, an incretinreceptor modulator, a DPP-4 inhibitor, or a PPAR agonist.

In another embodiment of the present invention, said anti-diabetic agentis metfomin, sitagliptin or ertuglifozin.

In another embodiment of the present invention, said anti-heart failureagent is an ACE inhibitor, an angiotensin receptor blocker, anangiotensin-receptor neprilysin inhibitor, a beta adrenergic receptorblocker, a calcium channel blocker, or a vasodilator.

While liver biopsy remains the standard for identification of NASHpatients, non-invasive methods for identifying patients withinflammatory liver disease have been described by Drescher, H., et al.,(“Current status in testing for nonalcoholic fatty liver disease (NAFLD)and non-alcoholic steatohepatitis (NASH), Cells 2019, 8, 845). Thesenon-invasive surrogate markers include, blood tests, liver functiontests, and imaging which have been successfully relied upon as a meansto identify inflammatory liver disease (hepatic steatosis,steatohepatitis, and fibrosis) and a measure for efficacy of a specifictherapy.

Hepatic steatosis (steatosis) is a key factor in NAFLD. While there isno specific serum marker existing today, there are several bloodbiomarkers panels that can be utilized to assess steatosis. These bloodbiomarkers may include, but are not limited to: i) NAFLD ridge score(parameters include ALT, HDL, cholesterol, triglycerides, HbA1c,leukocyte count hypertension); ii) NAFLD Liver Fat Score (NLFS)(parameters include liver fat content, metabolic syndrome, type-2diabetes, AST, AST:ALT, fasting insulin); iii) Hepatic Steatosis Index(HIS) (parameters include AST, ALT, BMI, diabetes, sex); iv) Fatty LiverIndex (FLI) (parameters include BMI, waist circumference, triglycerides,γ-glutamyl transferase); v) lipid accumulation product index (LAP)(parameters include sex, triglycerides, weight circumference); vi) FattyLiver Inhibition of Progression (FLIP) algorithm (parameters includehistological steatosis, disease activity, fibrosis score); vii) CHekscore (parameters include age, HbA1c, γ-glutamyl transferase,adiponectin, M30); viii) NAFLD Fibrosis Score (NFS) (parameters includeAST:ALT, albumin, platelet count, age, BMI, hyperglycemia); ix)Fibrosis-4-Score (FIB-4) (parameters include AST, ALT, platelet count,age); x) AST to Platelet Ratio Index (APRI) (parameters include AST,platelet count); xi) BARD Score (parameters include BMI, AST:ALT,diabetes); xii) Enhanced Liver Fibrosis panel (ELF) (parameters includeage, TIMP-1, PIIINP, hyaluronic acid); xiii) Hepascore (parametersinclude bilirubin, γ-glutamyl transferase, hyaluronic acid, α₂macroglobilin, age, gender); xiv) Fibro-Test-FibroSURE/Acti-Test(parameters include α₂ macroglobulin, haptoglobin, γ-glutamyltransferase, total bilirubin, apolipoprotein A1, ALT, age, gender); andxv) FibroMeter NAFLD index (parameters include platelet count,prothrombin index, ferritin, AST, ALT, body weight, age, liver stiffnessdetermined by vibration controlled transient elastography). Theparameters identified for each biomarker assist in the assessment ofliver damage/dysfunction (e.g., AST, ALT, γ-GT, platelet count,haptoglobin), lipid metabolism disorders (e.g., cholesterol,triglycerides), diabetes (e.g., HbA1c, fasting insulin level),inflammation (e.g., α₂ macroglobilin, ferritin).

Imaging techniques can also be used in conjunction with biopsy and bloodbiomarkers to identify NAFLD/NASH patients. Imaging techniques include,but are not limited to ultrasound (e.g., contrast-enhanced ultrasound(CEUS)); ultrasound-based elastography (e.g., vibration-controlledtransient elastography (VOTE; FibroScan), realtime shear waveelastography (SWE), acoustic radiation force impulse elastography(ARFI), supersonic shear imaging (SSI)); controlled attenuationparameters; magnetic resonance imaging (MRI) such as MRI proton densityfat fraction (MRI-PDFF); and magnetic resonance elastography (MRE).

In any of the present embodiments, the administration of the combinationin any of the above-mentioned therapeutically effective amounts can beadministered once or twice daily.

In any of the present embodiments, the administration of the combinationachieves a change in whole liver fat from baseline equal to or greaterthan about 30%.

In other instances, the administration of the combination achieves achange in whole liver fat from baseline equal to or greater than about50%.

In any of the present embodiments, identification of a patient may bethrough use of one or more blood marker panels. Suitable blood markerpanels include, but are not limited to the group consisting of NAFLDridge score, NAFLD Liver Fat Score (NLFS), Hepatic Steatosis Index(HIS), Fatty Liver Index (FLI), Lipid accumulation product index (LAP),Fatty Liver Inhibition of Progress (FLIP) algorithm, CHeK score, NALFDFibrosis Score (NFS), Fibrosis-4 Score (Fib-4), AST to Platelet RatioIndex (APRI), BARD score, Enhanced Liver Fibrosis panel (ELF),Hepascore, FibroTest-FibroSURE/ActiTest, ibroMeter NAFLD index, and anycombinations of the foregoing.

In certain embodiments, when a patient is identified as having hepaticsteatosis, the blood marker panel utilized is the NAFLD ridge score. Inanother embodiment, the blood marker panel is NAFLD Liver Fat Score(NLFS). In another embodiment, the blood marker panel is Fatty LiverIndex (FLI).

In certain embodiments, when the patient is identified as havingsteatohepatitis, the blood marker panel utilized is the Fatty LiverInhibition of Progress (FLIP) algorithm. In another embodiment, theblood marker panel is the CHeK score.

In certain embodiments, when a patient is identified as having fibrosis,the blood marker panel utilized is the NAFLD Fibrosis Score (NFS). Inanother embodiment, the blood marker panel is the Fibrosis-4 score(Fib-4). In another embodiment, the blood marker panel is the AST toPlatelet Ratio Index (APRI). In another embodiment, the blood markerpanel is the BARD score.

In certain other embodiments, in the methods described above, the stepof identifying a patient with hepatic steatosis, steatohepatitis orboth, further includes the use of imaging. The imaging may include, butis not limited to, ultrasound, ultrasound-based elastography, controlledattenuation parameter (CAP), magnetic resonance imaging (MRI), magneticresonance elastography, or a combination of the foregoing. In oneembodiment, the imaging is contrast-enhanced ultrasound (CEUS). Inanother embodiment, the imaging is ultrasound-based elastography isselected from vibration-controlled transient elastography (VCTE),acoustic radiation force impulse elastography (ARFI), supersonic shearimaging (SSI), or a combination of the foregoing. In another embodiment,the imaging is magnetic resonance imaging (MRI) or alternatively, MRIproton density fat fraction (MRI-PDFF). In another embodiment, theimaging is magnetic resonance elastography.

In addition to the above-mentioned methods and means for identifyinginflammatory liver disease in a patient, regulatory authority recognizedconditional approval for Phase III studies in NASH is based onhistological surrogate markers obtained by liver biopsy. These generallyaccepted surrogates are i) resolution of NASH without worsening offibrosis (i.e. a numerical increase in fibrosis stage); ii) a one ormore stage reduction in fibrosis without worsening of NASH. Details maybe found in: Ratziu, A critical review of endpoints for non-cirrhoticNASH therapeutic trials, Journal of Hepatology, 2018, 68. 353-361, andreferences therein.

Additionally, regulatory authorities look to a change in theNonalcoholic Fatty Liver Disease (NAFLD) Activity Score (NAS) frombaseline. The NAFLD Activity Score (NAS) is a composite score equal tothe sum of the steatosis grade (0-3), lobular inflammation grade (0-3),and hepatocellular ballooning grade (0-2), from centralized pathologistscoring of liver biopsies. The overall scale of the NAS is 0-8, withhigher scores indicating more severe disease. The outcome measure,change from baseline in NAFLD Activity Score (NAS), has a possible rangefrom −8 to +8, with negative values indicating a better outcome(improvement) and positive values indicating a worse outcome. Componentsof the NAS are scored as follows: Steatosis grade 0=<5% steatosis,1=5-33% steatosis, 2=34-66% steatosis, 3=>66% steatosis. Lobularinflammation grade=amount of lobular inflammation (combines mononuclear,fat granulomas, and polymorphonuclear (pmn) foci): 0=0, 1=<2 under 20×magnification, 2=2-4 under 20× magnification, 3=>4 under 20×magnification. Hepatocellular ballooning 0=none, 1=mild, 2=more thanmild.

In addition to the above-mentioned methods, regulatory authorityrecognized full approval for drugs to treat NASH is based ondemonstrating efficacy against one or more clinical measures including(1) progression to cirrhosis on histopathology, (2) reduction in hepaticdecompensation events (including hepatic encephalopathy, varicealbleeding, ascites), (3) change in MELD score from less than or equal to12 to more than 15, (4) liver transplant, or (5) all-cause mortality.

Combination Agents

The compounds of the present invention can be administered alone or incombination with one or more additional therapeutic agents. By“administered in combination” or “combination therapy” it is meant thata compound of the present invention and one or more additionaltherapeutic agents are administered concurrently to the mammal beingtreated. When administered in combination, each component may beadministered at the same time or sequentially in any order at differentpoints in time. Thus, each component may be administered separately butsufficiently closely in time so as to provide the desired therapeuticeffect. The phrases “concurrent administration,” “co-administration,”“simultaneous administration,” and “administered simultaneously” meanthat the compounds are administered in combination. Thus, the methods ofprevention and treatment described herein include use of combinationagents.

The combination agents are administered to a mammal in a therapeuticallyeffective amount. By “therapeutically effective amount” it is meant anamount of a compound of the present invention that, when administeredalone or in combination with an additional therapeutic agent to amammal, is effective to treat the desired disease/condition (e.g., NASH,heart failure or diabetes).

Given the NASH/NAFLD activity of the compounds of this invention, theymay be co-administered with other agents for the treatment ofnon-alcoholic steatohepatitis (NASH) and/or non-alcoholic fatty liverdisease (NAFLD) and associated disease/conditions, such as orlistat,TZDs and other insulin-sensitizing agents, FGF21 analogs, metformin,omega-3-acid ethyl esters (e.g. Lovaza), Fibrates, HMG-CoA reductaseinhibitors (e.g., pravastatin, lovastatin, atorvastatin, simvastatin,fluvastatin, NK-104 (a.k.a. itavastatin, or nisvastatin or nisbastatin)and ZD-4522 (a.k.a. rosuvastatin, or atavastatin or visastatin)),ezetimibe, probucol, ursodeoxycholic acid, TGR5 agonists, FXR agonists,Vitamin E, betaine, pentoxifylline, CB1 antagonists, carnitine,N-acetylcysteine, Reduced glutathione, lorcaserin, the combination ofnaltrexone with buproprion, SGLT2 inhibitors (including dapagliflozin,canagliflozin, empagliflozin, tofogliflozin, ertugliflozin, ASP-1941,THR1474, TS-071, ISIS388626 and LX4211 as well as those inWO2010023594), phentermine, topiramate, GLP-1 receptor agonists, GIPreceptor agonists, dual GLP-1 receptor/glucagon receptor agonists (i.e.,OPK88003, MEDI0382, JNJ-64565111, NN9277, BI 456906), dual GLP-1receprtor/GIP receptor agonists (i.e., Tirzepatide (LY3298176), NN9423),Angiotensin-receptor blockers an acetyl-CoA carboxylase (ACC) inhibitor,a diacylglycerol O-acyltransferase 1 (DGAT-1) inhibitor, such as thosedescribed in WO09016462 or WO2010086820, AZD7687 or LCQ908, adiacylglycerol O-acyltransferase 2 (DGAT-2) inhibitor, a PNPLA3inhibitor, an FGF21 analog, an FGF19 analog, a PPAR agonist, an FXRagonist, an AMPK activator, an SCD1 inhibitor or an MPO inhibitor.

Exemplary GLP-1 receptor agonists include liraglutide, albiglutide,exenatide, albiglutide, lixisenatide, dulaglutide, semaglutide, HM15211,LY3298176, Medi-0382, NN-9924, TTP-054, TTP-273, efpeglenatide, thosedescribed in WO2018109607, and those described in PCT/IB2019/054867filed Jun. 11, 2019 including the following:

-   2-({4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-7-fluoro-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-7-fluoro-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[2-(4-Cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[2-(5-Chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[2-(4-Chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-3-(1,3-oxazol-2-ylmethyl)-3H-imidazo[4,5-b]pyridine-5-carboxylic    acid;-   2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(1-ethyl-1H-imidazol-5-yl)methyl]-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(1,3-oxazol-4-ylmethyl)-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(pyridin-3-ylmethyl)-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(1,3-oxazol-5-ylmethyl)-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(1-ethyl-1H-1,2,3-triazol-5-yl)methyl]-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(1,3-oxazol-2-ylmethyl)-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[2-(4-chloro-2-fluorophenyl)-7-fluoro-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(1,3-oxazol-2-ylmethyl)-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-7-fluoro-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-7-fluoro-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[(2S)-2-(4-Cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[(2S)-2-(5-Chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(1-ethyl-1H-imidazol-5-yl)methyl]-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[(2R)-2-(4-Cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[(2R)-2-(5-Chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[(2R)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(1-ethyl-1H-imidazol-5-yl)methyl]-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[2-(5-Chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[(2S)-2-(5-Chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[(2R)-2-(5-Chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic    acid;-   2-({4-[2-(5-Chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic    acid, DIAST-X2; and-   2-[(4-{6-[(4-Cyano-2-fluorobenzyl)oxy]pyridin-2-yl}piperidin-1-yl)methyl]-1-[(2S)oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic    acid, or pharmaceutically acceptable salts thereof.

Exemplary ACC inhibitors include4-(4-[(1-isopropyl-7-oxo-1,4,6,7-tetrahydro-1′H-spiro[indazole-5,4′-piperidin]-1′-yl)carbonyl]-6-methoxypyridin-2-yl)benzoicacid; and firsocostat (GS-0976) and pharmaceutically acceptable saltsthereof.

Exemplary FXR Agonists include tropifexor(2-[(1R,3R,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylicacid); cilofexor (GS-9674); obeticholic acid; LY2562175, Met409,TERN-101; and EDP-305 and pharmaceutically acceptable salts thereof.

Exemplary DGAT2 inhibitors include(S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide;

-   2-(5-((3-ethoxy-5-fluoropyridin-2-yl)oxy)pyridin-3-yl)-N-((3R,4S)-4-fluoropiperidin-3-yl)pyrimidine-5-carboxamide;-   2-(5-((3-ethoxy-5-fluoropyridin-2-yl)oxy)pyridin-3-yl)-N-((3S,5S)-5-fluoropiperidin-3-yl)pyrimidine-5-carboxamide;-   2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-((3R,4S)-4-fluoropiperidin-3-yl)pyrimidine-5-carboxamide;-   2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-((3R,4R)-4-fluoropiperidin-3-yl)pyrimidine-5-carboxamide;-   2-(5-((3-ethoxy-5-fluoropyridin-2-yl)oxy)pyridin-3-yl)-N-((3R,4R)-4-fluoropiperidin-3-yl)pyrimidine-5-carboxamide;    and-   2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-((3S,5S)-5-fluoropiperidin-3-yl)pyrimidine-5-carboxamide,    and pharmaceutically acceptable salts thereof.

Exemplary KHK inhibitors include[(1R,5S,6R)-3-{2-[(2S)-2-methylazetidin-1-yl]-6-(trifluoromethyl)pyrimidin-4-yl}-3-azabicyclo[3.1.0]hex-6-yl]aceticacid and pharmaceutically acceptable salts thereof.

Given the anti-diabetic activity of the compounds of this invention theymay be co-administered with other anti-diabetic agents. Suitableanti-diabetic agents include insulin, metformin, GLP-1 receptor agonists(described herein above), an acetyl-CoA carboxylase (ACC) inhibitor(described herein above), SGLT2 inhibitors (described herein above),monoacylglycerol O-acyltransferase inhibitors, phosphodiesterase(PDE)-10 inhibitors, AMPK activators, sulfonylureas (e.g.,acetohexamide, chlorpropamide, diabinese, glibenclamide, glipizide,glyburide, glimepiride, gliclazide, glipentide, gliquidone, glisolamide,tolazamide, and tolbutamide), meglitinides, aamylase inhibitors (e.g.,tendamistat, trestatin and AL-3688), an α-glucoside hydrolase inhibitor(e.g., acarbose), α-glucosidase inhibitors (e.g., adiposine,camiglibose, emiglitate, miglitol, voglibose, pradimicin-Q, andsalbostatin), PPARy agonists (e.g., balaglitazone, ciglitazone,darglitazone, englitazone, isaglitazone, pioglitazone androsiglitazone), PPAR α/γ agonists (e.g., CLX-0940, GW-1536, GW-1929,GW-2433, KRP-297, L-796449, LR-90, MK-0767 and SB-219994), proteintyrosine phosphatase-1B (PTP-1 B) inhibitors (e.g., trodusquemine,hyrtiosal extract, and compounds disclosed by Zhang, S., et al., DrugDiscovery Today, 12(9/10), 373-381 (2007)), SIRT-1 activators (e.g.,resveratrol, GSK2245840 or GSK184072), dipeptidyl peptidease IV (DPP-IV)inhibitors (e.g., those in WO2005116014, sitagliptin, vildagliptin,alogliptin, dutogliptin, linagliptin and saxagliptin), insulinsecreatagogues, a fatty acid oxidation inhibitors, A2 antagonists, c-junamino-terminal kinase (JNK) inhibitors, glucokinase activators (GKa)such as those described in WO2010103437, WO2010103438, WO2010013161,WO2007122482, TTP-399, TTP-355, TTP-547, AZD1656, ARRY403, MK-0599,TAK-329, AZD5658 or GKM-001, insulin, insulin mimetics, glycogenphosphorylase inhibitors (e.g. GSK1362885), VPAC2 receptor agonists,glucagon receptor modulators such as those described in Demong, D. E. etal. Annual Reports in Medicinal Chemistry 2008, 43, 119-137, GPR119modulators, particularly agonists, such as those described inWO2010140092, WO2010128425, WO2010128414, WO2010106457, Jones, R. M. etal. in Medicinal Chemistry 2009, 44, 149-170 (e.g. MBX-2982, GSK1292263,APD597 and PSN821), FGF21 derivatives or analogs such as those describedin Kharitonenkov, A. et al. et al., Current Opinion in InvestigationalDrugs 2009, 10(4)359-364, TGR5 (also termed GPBAR1) receptor modulators,particularly agonists, such as those described in Zhong, M., CurrentTopics in Medicinal Chemistry, 2010, 10(4), 386-396 and INT777, GPR40agonists, such as those described in Medina, J. C., Annual Reports inMedicinal Chemistry, 2008, 43, 75-85, including but not limited toTAK-875, GPR120 modulators, particularly agonists, high affinitynicotinic acid receptor (HM74A) activators, and SGLT1 inhibitors, suchas GSK1614235. A further representative listing of anti-diabetic agentsthat can be combined with the compounds of the present invention can befound, for example, at page 28, line 35 through page 30, line 19 ofWO2011005611.

Other antidiabetic agents could include inhibitors or modulators ofcarnitine palmitoyl transferase enzymes, inhibitors of fructose1,6-diphosphatase, inhibitors of aldose reductase, mineralocorticoidreceptor inhibitors, inhibitors of TORC2, inhibitors of CCR2 and/orCCR5, inhibitors of PKC isoforms (e.g. PKCα, PKCβ, PKCγ), inhibitors offatty acid synthetase, inhibitors of serine palmitoyl transferase,modulators of GPR81, GPR39, GPR43, GPR41, GPR105, Kv1.3, retinol bindingprotein 4, glucocorticoid receptor, somatostain receptors (e.g. SSTR1,SSTR2, SSTR3 and SSTRS), inhibitors or modulators of PDHK2 or PDHK4,inhibitors of MAP4K4, modulators of IL1 family including IL1 beta,modulators of RXRalpha. In addition suitable anti-diabetic agentsinclude mechanisms listed by Carpino, P. A., Goodwin, B. Expert Opin.Ther. Pat, 2010, 20(12), 1627-51.

Given the anti-heart failure activity of the compounds of the presentinvention they may be co-administered with other anti-heart failureagents such as ACE inhibitors (e.g. benzepril, zofenopril, captopril,enalapril, fosinopril, lisinopril, perindopril, quinapril, ramipril,trandolapril), Angiotensin II receptor blockers (e.g., candesartan,losartan, valsartan), Angiotensin-receptor blocker/neprilysin inhibitor(sacubitril/valsartan), If channel blocker Ivabradine, Beta-Adrenergicblocking agents (e.g., bisoprolol, metoprolol succinate, carvedilol),Aldosterone antagonists (e.g., spironolactone, eplerenone), hydralazineand isosorbide dinitrate, diuretics (e.g., furosemide, bumetanide,torsemide, chlorothiazide, amiloride, hydrochlorothiazide, Indapamide,Metolazone, Triamterene), or digoxin.

The compounds of the present invention may also be used in combinationwith antihypertensive agents and such antihypertensive activity isreadily determined by those skilled in the art according to standardassays (e.g., blood pressure measurements). Examples of suitableanti-hypertensive agents include: alpha adrenergic blockers; betaadrenergic blockers; calcium channel blockers (e.g., diltiazem,verapamil, nifedipine and amlodipine); vasodilators (e.g., hydralazine),diruetics (e.g., chlorothiazide, hydrochlorothiazide, flumethiazide,hydroflumethiazide, bendroflumethiazide, methylchlorothiazide,trichloromethiazide, polythiazide, benzthiazide, ethacrynic acidtricrynafen, chlorthalidone, torsemide, furosemide, musolimine,bumetanide, triamtrenene, amiloride, spironolactone); renin inhibitors;ACE inhibitors (e.g., captopril, zofenopril, fosinopril, enalapril,ceranopril, cilazopril, delapril, pentopril, quinapril, ramipril,lisinopril); AT-1 receptor antagonists (e.g., losartan, irbesartan,valsartan); ET receptor antagonists (e.g., sitaxsentan, atrsentan andcompounds disclosed in U.S. Pat. Nos. 5,612,359 and 6,043,265); DualET/All antagonist (e.g., compounds disclosed in WO 00/01389); neutralendopeptidase (NEP) inhibitors; vasopepsidase inhibitors (dual NEP-ACEinhibitors) (e.g., gemopatrilat and nitrates). An exemplary antianginalagent is ivabradine.

Examples of suitable calcium channel blockers (L-type or T-type) includediltiazem, verapamil, nifedipine and amlodipine and mybefradil.

Examples of suitable cardiac glycosides include digitalis and ouabain.

In one embodiment, a Formula I compound may be co-administered with oneor more diuretics. Examples of suitable diuretics include (a) loopdiuretics such as furosemide (such as LASIX™), torsemide (such asDEMADEX™), bemetanide (such as BUMEX™), and ethacrynic acid (such asEDECRIN™), (b) thiazide-type diuretics such as chlorothiazide (such asDIURIL™, ESIDRIX™ or HYDRODIURIL™) hydrochlorothiazide (such asMICROZIDE™ or ORETIC™), benzthiazide, hydroflumethiazide (such asSALURON™), bendroflumethiazide, methychlorthiazide, polythiazide,trichlormethiazide, and indapamide (such as LOZOL™), (c)phthalimidine-type diuretics such as chlorthalidone (such as HYGROTON™),and metolazone (such as ZAROXOLYN™), (d) quinazoline-type diuretics suchas quinethazone; and (e) potassium-sparing diuretics such as triamterene(such as DYRENIUM™), and amiloride (such as MIDAMOR™ or MODURETIC™).

In another embodiment, a compound of Formula I may be co-administeredwith a loop diuretic. In still another embodiment, the loop diuretic isselected from furosemide and torsemide. In still another embodiment, oneor more compounds of Formula I may be co-administered with furosemide.In still another embodiment, one or more compounds of Formula I may beco-administered with torsemide which may optionally be a controlled ormodified release form of torsemide.

In another embodiment, a compound of Formula I may be co-administeredwith a thiazide-type diuretic. In still another embodiment, thethiazide-type diuretic is selected from the group consisting ofchlorothiazide and hydrochlorothiazide. In still another embodiment, oneor more compounds of Formula I may be co-administered withchlorothiazide. In still another embodiment, one or more compounds ofFormula I may be co-administered with hydrochlorothiazide.

In another embodiment, one or more compounds of Formula I may beco-administered with a phthalimidine-type diuretic. In still anotherembodiment, the phthalimidine-type diuretic is chlorthalidone.

Examples of suitable mineralocorticoid receptor antagonists includesprionolactone and eplerenone.

Examples of suitable phosphodiesterase inhibitors include: PDE IIIinhibitors (such as cilostazol); and PDE V inhibitors (such assildenafil).

Those skilled in the art will recognize that the compounds of thisinvention may also be used in conjunction with other cardiovascular orcerebrovascular treatments including PCI, stenting, drug-eluting stents,stem cell therapy and medical devices such as implanted pacemakers,defibrillators, or cardiac resynchronization therapy.

Particularly when provided as a single dosage unit, the potential existsfor a chemical interaction between the combined active ingredients. Forthis reason, when a Formula I compound and a second therapeutic agentare combined in a single dosage unit they are formulated such thatalthough the active ingredients are combined in a single dosage unit,the physical contact between the active ingredients is minimized (thatis, reduced). For example, one active ingredient may be enteric coated.By enteric coating one of the active ingredients, it is possible notonly to minimize the contact between the combined active ingredients,but also, it is possible to control the release of one of thesecomponents in the gastrointestinal tract such that one of thesecomponents is not released in the stomach but rather is released in theintestines. One of the active ingredients may also be coated with amaterial that effects a sustained release throughout thegastrointestinal tract and also serves to minimize physical contactbetween the combined active ingredients. Furthermore, thesustained-released component can be additionally enteric coated suchthat the release of this component occurs only in the intestine. Stillanother approach would involve the formulation of a combination productin which the one component is coated with a sustained and/or entericrelease polymer, and the other component is also coated with a polymersuch as a low viscosity grade of hydroxypropyl methylcellulose (HPMC) orother appropriate materials as known in the art, in order to furtherseparate the active components. The polymer coating serves to form anadditional barrier to interaction with the other component.

These as well as other ways of minimizing contact between the componentsof combination products of the present invention, whether administeredin a single dosage form or administered in separate forms but at thesame time by the same manner, will be readily apparent to those skilledin the art, once armed with the present disclosure.

In combination therapy treatment, both the compounds of this inventionand the other drug therapies are administered to mammals (e.g., humans,male or female) by conventional methods.

The Formula I compounds of this invention, their prodrugs and the saltsof such compounds and prodrugs are all adapted to therapeutic use asagents that inhibit and or degrade BCKDK in mammals, particularly humansand thus are useful for the treatment of the various conditions (e.g.,those described herein) in which such action is implicated.

The disease/conditions that can be treated in accordance with thepresent invention include, but are not limited to NASH/NAFLD, diabetes,and heart failure and associated disease/conditions.

In particular, inhibition and/or degradation of BCKDK is associated withNASH/NAFLD and associated disease/conditions because Increased BCAAlevels were observed in human NASH samples (Lake A D, Novak P, ShipkovaP, Aranibar N, Robertson D G, Reily M D, Lehman-McKeeman L D,Vaillancourt R R, Cherrington N J: Branched chain amino acid metabolismprofiles in progressive human nonalcoholic fatty liver disease. AminoAcids 2015, 47:603-15). Reduced levels of PPM1K mRNA and increased BCKDKprotein levels were also observed in human NASH (Lake A D, Novak P,Shipkova P, Aranibar N, Robertson D G, Reily M D, Lehman-McKeeman L D,Vaillancourt R R, Cherrington N J: Branched chain amino acid metabolismprofiles in progressive human nonalcoholic fatty liver disease. AminoAcids 2015, 47:603-15). Treatment of obese mice or rats with a BCKDKinhibitor reduced hepatic steatosis and triglyceride content, andoverexpression of PPM1K in rats reduced hepatic triglyceride content(White P J, McGarrah R W, Grimsrud P A, Tso S C, Yang W H, Haldeman J M,Grenier-Larouche T, An J, Lapworth A L, Astapova I, Hannou S A, GeorgeT, Arlotto M, Olson L B, Lai M, Zhang G F, Ilkayeva O, Herman M A, WynnR M, Chuang D T, Newgard C B: The BCKDH Kinase and Phosphatase IntegrateBCAA and Lipid Metabolism via Regulation of ATP-Citrate Lyase. CellMetab 2018, 27(6), 1281-1293).

Further, regulatory authority recognized conditional approval for PhaseIII studies in NASH is based on histological surrogate markers obtainedby liver biopsy. These generally accepted surrogates are i) resolutionof NASH without worsening of fibrosis (i.e. a numerical increase infibrosis stage); ii) a one or more stage reduction in fibrosis withoutworsening of NASH. Details may be found in: Ratziu, A critical review ofendpoints for non-cirrhotic NASH therapeutic trials, Journal ofHepatology, 2018, 68. 353-361, and references therein.

Accordingly, given the positive correlation between activation of BCKDKwith the development of NASH/NAFLD and associated disease/conditions,Formula I compounds of this invention, their prodrugs and the salts ofsuch compounds and prodrugs, by virtue of their pharmacologic action,are useful for the prevention, arrestment and/or regression of fattyliver, nonalcoholic fatty liver disease, non-alcoholic steatohepatitis,nonalcoholic steatohepatitis with liver fibrosis, non-alcoholicsteatohepotitis with cirrhosis, nonalcoholic steatohepatitis withcirrhosis and hepatocellular carcinoma and nonalcoholic steatohepatitiswith cirrhosis and with a metabolic-related disease.

Similarly, Formula I compounds of this invention, their prodrugs and thesalts of such compounds and prodrugs, by virtue of their pharmacologicaction, are useful for the prevention, arrestment and/or regression ofalcoholic fatty liver disease, alcoholic steatohepatitis, alcoholicsteatohepatitis with liver fibrosis, alcoholic steatohepatitis withcirrhosis, alcoholic steatohepatitis with cirrhosis and withhepatocellular carcinoma, and alcoholic steatohepatitis with cirrhosisand with a metabolic-related disease.

In addition, increased BCKDK is associated with heart failure andassociated disease/conditions because an increase in BCKA have beenobserved in hearts from patients with heart failure. (Sun H, Olson K C,Gao C, Prosdocimo D A, Zhou M, Wang Z, Jeyaraj D, Youn J Y, Ren S, LiuY, Rau C D, Shah S, Ilkayeva O, Gui W J, William N S, Wynn R M, NewgardC B, Cai H, Xiao X, Chuang D T, Schulze P C, Lynch C, Jain M K, Wang Y:Catabolic Defect of Branched-Chain Amino Acids Promotes Heart Failure.Circulation 2016, 133:2038-49.)

In heart failure, the regulatory phosphatase that activates BCKDH(PPM1K) is downregulated, and BCKDK is upregulated; thus BCAA catabolismis likely impaired in heart failure. (Sun H, Olson K C, Gao C,Prosdocimo D A, Zhou M, Wang Z, Jeyaraj D, Youn J Y, Ren S, Liu Y, Rau CD, Shah S, Ilkayeva O, Gui W J, William N S, Wynn R M, Newgard C B, CaiH, Xiao X, Chuang D T, Schulze P C, Lynch C, Jain M K, Wang Y: CatabolicDefect of Branched-Chain Amino Acids Promotes Heart Failure. Circulation2016, 133:2038-49.)

Both BCKDH and BCKDK are expressed ubiquitously; however, the regulatoryphosphatase PPM1K, which dephosphorylates BCKDH, is expressed mosthighly in cardiac tissue. Mice lacking PPM1K develop aging-induced heartfailure and have worsened heart function when subjected to a transverseaortic constriction (TAC) heart failure model. (Sun H, Olson K C, Gao C,Prosdocimo D A, Zhou M, Wang Z, Jeyaraj D, Youn J Y, Ren S, Liu Y, Rau CD, Shah S, Ilkayeva O, Gui W J, William N S, Wynn R M, Newgard C B, CaiH, Xiao X, Chuang D T, Schulze P C, Lynch C, Jain M K, Wang Y: CatabolicDefect of Branched-Chain Amino Acids Promotes Heart Failure. Circulation2016, 133:2038-49.)

Use of an inhibitor of BCKDK improved cardiac function in threedifferent preclinical heart failure models (TAC, left anteriordescending artery ligation/myocardial infarct, andischemia/reperfusion). (Sun H, Olson K C, Gao C, Prosdocimo D A, Zhou M,Wang Z, Jeyaraj D, Youn J Y, Ren S, Liu Y, Rau C D, Shah S, Ilkayeva O,Gui W J, William N S, Wynn R M, Newgard C B, Cai H, Xiao X, Chuang D T,Schulze P C, Lynch C, Jain M K, Wang Y: Catabolic Defect ofBranched-Chain Amino Acids Promotes Heart Failure. Circulation 2016,133:2038-49; Wang W, Zhang F, Xia Y, Zhao S, Yan W, Wang H, Lee Y, Li C,Zhang L, Lian K, Gao E, Cheng H, Tao L: Defective branched chain aminoacid catabolism contributes to cardiac dysfunction and remodelingfollowing myocardial infarction. Am J Physiol Heart Circ Physiol 2016,311:H1160-H9; Li T, Zhang Z, Kolwicz S C, Jr., Abell L, Roe N D, Kim M,Zhou B, Cao Y, Ritterhoff J, Gu H, Raftery D, Sun H, Tian R: DefectiveBranched-Chain Amino Acid Catabolism Disrupts Glucose Metabolism andSensitizes the Heart to lschemia-Reperfusion Injury. Cell Metab 2017,25:374-85.)

Therefore, inhibiting/degrading BCKDK in cardiac or peripheral tissueshould demonstrate benefit for metabolic disease and cardiac function.

Accordingly, given the positive correlation between activation of BCKDKwith the development of heart failure and associated disease/conditions,Formula I compounds of this invention, their prodrugs and the salts ofsuch compounds and prodrugs, by virtue of their pharmacologic action,are useful for the prevention, arrestment and/or regression of heartfailure, congestive heart failure, unstable angina, peripheral arterialdisease, pulmonary hypertension, vasculitis or where the mammal hasexperienced myocardial infarction (secondary prevention (2^(nd)myocardial infarction)).

In addition, increased BCKDK is associated with diabetes and associateddisease/conditions because plasma BCAA are upregulated in patients withincreased fasting glucose levels, and a one standard deviation increasein BCKA concentrations in plasma increases the likelihood of developingdiabetes by over 50%. (Wang T J, Larson M G, Vasan R S, Cheng S, Rhee EP, McCabe E, Lewis G D, Fox C S, Jacques P F, Fernandez C, O'Donnell CJ, Carr S A, Mootha V K, Florez J C, Souza A, Melander O, Clish C B,Gerszten R E: Metabolite profiles and the risk of developing diabetes.Nat Med 2011, 17:448-53; Newgard C B, An J, Bain J R, Muehlbauer M J,Stevens R D, Lien L F, Haqq A M, Shah S H, Arlotto M, Slentz C A, RochonJ, Gallup D, Ilkayeva O, Wenner B R, Yancy W S, Jr., Eisenson H, MusanteG, Surwit R S, Millington D S, Butler M D, Svetkey L P: A branched-chainamino acid-related metabolic signature that differentiates obese andlean humans and contributes to insulin resistance. Cell Metab 2009,9:311-26; Menni C, Fauman E, Erte I, Perry J R, Kastenmuller G, Shin SY, Petersen A K, Hyde C, Psatha M, Ward K J, Yuan W, Milburn M, Palmer CN, Frayling™, Trimmer J, Bell J T, Gieger C, Mohney R P, Brosnan M J,Suhre K, Soranzo N, Spector T D: Biomarkers for type 2 diabetes andimpaired fasting glucose using a nontargeted metabolomics approach.Diabetes 2013, 62:4270-6.)

Genetic analyses suggest that loss of function mutations in the PPM1Klocus increase BCAA/BCKA levels and are associated with development oftype 2 diabetes. (Lotta L A, Scott R A, Sharp S J, Burgess S, Luan J,Tillin T, Schmidt A F, Imamura F, Stewart I D, Perry J R, Marney L,Koulman A, Karoly E D, Forouhi N G, Sjogren R J, Naslund E, Zierath J R,Krook A, Savage D B, Griffin J L, Chaturvedi N, Hingorani A D, Khaw K T,Barroso I, McCarthy M I, O'Rahilly S, Wareham N J, Langenberg C: GeneticPredisposition to an Impaired Metabolism of the Branched-Chain AminoAcids and Risk of Type 2 Diabetes: A Mendelian Randomisation Analysis.PLoS Med 2016, 13:e1002179.)

Treatment of diabetic, obese mice or rats with a BCKDK inhibitorimproved fasting glycemia, glycemia in a glucose tolerance test, reducedinsulin levels, and improved insulin sensitivity. Overexpression ofPPM1K in rats also improved glycemia and reduced insulin levels. (WhiteP J, McGarrah R W, Grimsrud P A, Tso S C, Yang W H, Haldeman J M,Grenier-Larouche T, An J, Lapworth A L, Astapova I, Hannou S A, GeorgeT, Arlotto M, Olson L B, Lai M, Zhang G F, Ilkayeva O, Herman M A, WynnR M, Chuang D T, Newgard C B: The BCKDH Kinase and Phosphatase IntegrateBCAA and Lipid Metabolism via Regulation of ATP-Citrate Lyase. CellMetab 2018, 27 (60, 1281-1293.)

Accordingly, given the positive correlation between BCKDK and thedevelopment of diabetes and associated disease/conditions, Formula Icompounds of this invention, their prodrugs and the salts of suchcompounds and prodrugs, by virtue of their pharmacologic action, areuseful for the prevention, arrestment and/or regression of Type Idiabetes, Type II diabetes mellitus, idiopathic Type I diabetes (TypeIb), latent autoimmune diabetes in adults (LADA), early-onset Type 2diabetes (EOD), youth-onset atypical diabetes (YOAD), maturity onsetdiabetes of the young (MODY), malnutrition-related diabetes, gestationaldiabetes, coronary heart disease, ischemic stroke, restenosis afterangioplasty, peripheral vascular disease, intermittent claudication,myocardial infarction, dyslipidemia, post-prandial lipemia, conditionsof impaired glucose tolerance (IGT), conditions of impaired fastingplasma glucose, metabolic acidosis, ketosis, arthritis, diabeticretinopathy, macular degeneration, cataract, diabetic nephropathy,glomerulosclerosis, chronic renal failure, diabetic neuropathy,metabolic syndrome, syndrome X, hyperglycemia, hyperinsulinemia,hypertrygliceridemia, insulin resistance, impaired glucose metabolism,skin and connective tissue disorders, foot ulcerations and ulcerativecolitis, endothelial dysfunction and impaired vascular compliance, andhyper apo B lipoproteinemia.

The utility of the Formula I compounds of the invention, their prodrugsand the salts of such compounds and prodrugs as medical agents in thetreatment of the above described disease/conditions in mammals (e.g.humans, male or female) is demonstrated by the activity of the compoundsof this invention in conventional in vitro and in vivo assays describedbelow. The in vivo assays (with appropriate modifications within theskill in the art) may be used to determine the activity of other agentsas well as the compounds of this invention. Such assays also provide ameans whereby the activities of the Formula I compounds of thisinvention, their prodrugs and the salts of such compounds and prodrugs(or the other agents described herein) can be compared to each other andwith the activities of other known compounds. The results of thesecomparisons are useful for determining dosage levels in mammals,including humans, for the treatment of such diseases.

Administration of the compounds of this invention can be via any methodwhich delivers a compound of this invention systemically and/or locally.These methods include oral routes, parenteral, intraduodenal routes,buccal, intranasal etc. Generally, the compounds of this invention areadministered orally, but parenteral administration (e.g., intravenous,intramuscular, subcutaneous or intramedullary) may be utilized, forexample, where oral administration is inappropriate for the target orwhere the patient is unable to ingest the drug.

For administration to human patients, an oral daily dose of thecompounds herein may be in the range 1 mg to 5000 mg depending, ofcourse, on the mode of and frequency of administration, the diseasestate, and the age and condition of the patient, etc. An oral daily doseis in the range of 3 mg to 2000 mg may be used. A further oral dailydose is in the range of 5 mg to 1000 mg. For convenience, the compoundsof the present invention can be administered in a unit dosage form. Ifdesired, multiple doses per day of the unit dosage form can be used toincrease the total daily dose. The unit dosage form, for example, may bea tablet or capsule containing about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175,200, 250, 500, or 1000 mg of the compound of the present invention. Thetotal daily dose may be administered in single or divided doses and may,at the physician's discretion, fall outside of the typical ranges givenherein.

For administration to human patients, an infusion daily dose of thecompounds herein may be in the range 1 mg to 2000 mg depending, ofcourse, on the mode of and frequency of administration, the diseasestate, and the age and condition of the patient, etc. A further infusiondaily dose is in the range of 5 mg to 1000 mg. The total daily dose maybe administered in single or divided doses and may, at the physician'sdiscretion, fall outside of the typical ranges given herein.

These compounds may also be administered to animals other than humans,for example, for the indications detailed above. The precise dosageadministered of each active ingredient will vary depending upon anynumber of factors, including but not limited to, the type of animal andtype of disease state being treated, the age of the animal, and theroute(s) of administration.

A dosage of the combination pharmaceutical agents to be used inconjuction with the Formula I compounds is used that is effective forthe indication being treated. Such dosages can be determined by standardassays such as those referenced above and provided herein. Thecombination agents may be administered simultaneously or sequentially inany order.

These dosages are based on an average human subject having a weight ofabout 60 kg to 70 kg. The physician will readily be able to determinedoses for subjects whose weight falls outside this range, such asinfants and the elderly.

Dosage regimens may be adjusted to provide the optimum desired response.For example, a single bolus may be administered, several divided dosesmay be administered over time or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation. It is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form, as used herein, refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the chemotherapeuticagent and the particular therapeutic or prophylactic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

Thus, the skilled artisan would appreciate, based upon the disclosureprovided herein, that the dose and dosing regimen is adjusted inaccordance with methods well-known in the therapeutic arts. That is, themaximum tolerable dose can be readily established, and the effectiveamount providing a detectable therapeutic benefit to a patient may alsobe determined, as can the temporal requirements for administering eachagent to provide a detectable therapeutic benefit to the patient.Accordingly, while certain dose and administration regimens areexemplified herein, these examples in no way limit the dose andadministration regimen that may be provided to a patient in practicingthe present invention.

It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated, and may include single or multipledoses. It is to be further understood that for any particular subject,specific dosage regimens should be adjusted over time according to theindividual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition. Forexample, doses may be adjusted based on pharmacokinetic orpharmacodynamic parameters, which may include clinical effects such astoxic effects and/or laboratory values. Thus, the present inventionencompasses intra-patient dose-escalation as determined by the skilledartisan. Determining appropriate dosages and regiments foradministration of the chemotherapeutic agent are well-known in therelevant art and would be understood to be encompassed by the skilledartisan once provided the teachings disclosed herein.

The present invention further comprises use of a compound of Formula Ifor use as a medicament (such as a unit dosage tablet or unit dosagecapsule). In another embodiment, the present invention comprises the useof a compound of Formula I for the manufacture of a medicament (such asa unit dosage tablet or unit dosage capsule) to treat one or more of theconditions previously identified in the above sections discussingmethods of treatment.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The compounds of the invention or combinations can be administered alonebut will generally be administered in an admixture with one or moresuitable pharmaceutical excipients, adjuvants, diluents or carriersknown in the art and selected with regard to the intended route ofadministration and standard pharmaceutical practice. The compound of theinvention or combination may be formulated to provide immediate-,delayed-, modified-, sustained-, pulsed- or controlled-release dosageforms depending on the desired route of administration and thespecificity of release profile, commensurate with therapeutic needs.

The pharmaceutical composition comprises a compound of the invention ora combination in an amount generally in the range of from about 1% toabout 75%, 80%, 85%, 90% or even 95% (by weight) of the composition,usually in the range of about 1%, 2% or 3% to about 50%, 60% or 70%,more frequently in the range of about 1%, 2% or 3% to less than 50% suchas about 25%, 30% or 35%.

Methods of preparing various pharmaceutical compositions with a specificamount of active compound are known to those skilled in this art. Forexamples, see Remington: The Practice of Pharmacy, Lippincott Williamsand Wilkins, Baltimore Md. 20.sup.th ed. 2000.

Compositions suitable for parenteral injection generally includepharmaceutically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions, or emulsions, and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers or diluents(including solvents and vehicles) include water, ethanol, polyols(propylene glycol, polyethylene glycol, glycerol, and the like),suitable mixtures thereof, triglycerides including vegetable oils suchas olive oil, and injectable organic esters such as ethyl oleate. Apreferred carrier is Miglyol® brand caprylic/capric acid ester withglycerine or propylene glycol (e.g., Miglyol® 812, Miglyol® 829,Miglyol® 840) available from Condea Vista Co., Cranford, N.J. Properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants.

These compositions for parenteral injection may also contain excipientssuch as preserving, wetting, emulsifying, and dispersing agents.Prevention of microorganism contamination of the compositions can beaccomplished with various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, and the like. Itmay also be desirable to include isotonic agents, for example, sugars,sodium chloride, and the like. Prolonged absorption of injectablepharmaceutical compositions can be brought about by the use of agentscapable of delaying absorption, for example, aluminum monostearate andgelatin.

Solid dosage forms for oral administration include capsules, tablets,chews, lozenges, pills, powders, and multi-particulate preparations(granules). In such solid dosage forms, a compound of the presentinvention or a combination is admixed with at least one inert excipient,diluent or carrier. Suitable excipients, diluents or carriers includematerials such as sodium citrate or dicalcium phosphate and/or (a) oneor more fillers or extenders (e.g., microcrystalline cellulose(available as Avicel™ from FMC Corp.) starches, lactose, sucrose,mannitol, silicic acid, xylitol, sorbitol, dextrose, calcium hydrogenphosphate, dextrin, alpha-cyclodextrin, beta-cyclodextrin, polyethyleneglycol, medium chain fatty acids, titanium oxide, magnesium oxide,aluminum oxide and the like); (b) one or more binders (e.g.,carboxymethylcellulose, methylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, gelatin, gum arabic, ethyl cellulose,polyvinyl alcohol, pullulan, pregelatinized starch, agar, tragacanth,alginates, gelatin, polyvinylpyrrolidone, sucrose, acacia and the like);(c) one or more humectants (e.g., glycerol and the like); (d) one ormore disintegrating agents (e.g., agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain complex silicates, sodiumcarbonate, sodium lauryl sulphate, sodium starch glycolate (available asExplotab™ from Edward Mendell Co.), cross-linked polyvinyl pyrrolidone,croscarmellose sodium A-type (available as Ac-di-sol™), polyacrilinpotassium (an ion exchange resin) and the like); (e) one or moresolution retarders (e.g., paraffin and the like); (f) one or moreabsorption accelerators (e.g., quaternary ammonium compounds and thelike); (g) one or more wetting agents (e.g., cetyl alcohol, glycerolmonostearate and the like); (h) one or more adsorbents (e.g., kaolin,bentonite and the like); and/or (i) one or more lubricants (e.g., talc,calcium stearate, magnesium stearate, stearic acid, polyoxyl stearate,cetanol, talc, hydrogenated caster oil, sucrose esters of fatty acid,dimethylpolysiloxane, microcrystalline wax, yellow beeswax, whitebeeswax, solid polyethylene glycols, sodium lauryl sulfate and thelike). In the case of capsules and tablets, the dosage forms may alsocomprise buffering agents.

Solid compositions of a similar type may also be used as fillers in softor hard filled gelatin capsules using such excipients as lactose or milksugar, as well as high molecular weight polyethylene glycols, and thelike.

Solid dosage forms such as tablets, dragees, capsules, and granules maybe prepared with coatings and shells, such as enteric coatings andothers well known in the art. They may also contain opacifying agents,and can also be of such composition that they release the compound ofthe present invention and/or the additional pharmaceutical agent in adelayed manner. Examples of embedding compositions that can be used arepolymeric substances and waxes. The drug may also be inmicroencapsulated form, if appropriate, with one or more of theabove-mentioned excipients.

For tablets, the active agent will typically comprise less than 50% (byweight) of the formulation, for example less than about 10% such as 5%or 2.5% by weight. The predominant portion of the formulation comprisesfillers, diluents, disintegrants, lubricants and optionally, flavors.The composition of these excipients is well known in the art.Frequently, the fillers/diluents will comprise mixtures of two or moreof the following components: microcrystalline cellulose, mannitol,lactose (all types), starch, and di-calcium phosphate. Thefiller/diluent mixtures typically comprise less than 98% of theformulation and preferably less than 95%, for example 93.5%. Preferreddisintegrants include Ac-di-sol™, Explotab™, starch and sodium laurylsulphate. When present a disintegrant will usually comprise less than10% of the formulation or less than 5%, for example about 3%. Apreferred lubricant is magnesium stearate. When present a lubricant willusually comprise less than 5% of the formulation or less than 3%, forexample about 1%.

Tablets may be manufactured by standard tabletting processes, forexample, direct compression or a wet, dry or melt granulation, meltcongealing process and extrusion. The tablet cores may be mono ormulti-layer(s) and can be coated with appropriate overcoats known in theart.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Inaddition to the compound of the present invention or the combination,the liquid dosage form may contain inert diluents commonly used in theart, such as water or other solvents, solubilizing agents andemulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseedoil, groundnut oil, corn germ oil, olive oil, castor oil, sesame seedoil and the like), Miglyole® (available from CONDEA Vista Co., Cranford,N.J.), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols andfatty acid esters of sorbitan, or mixtures of these substances, and thelike.

Besides such inert diluents, the composition may also includeexcipients, such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, and perfuming agents.

Oral liquid forms of the compounds of the invention or combinationsinclude solutions, wherein the active compound is fully dissolved.Examples of solvents include all pharmaceutically precedented solventssuitable for oral administration, particularly those in which thecompounds of the invention show good solubility, e.g., polyethyleneglycol, polypropylene glycol, edible oils and glyceryl- andglyceride-based systems. Glyceryl- and glyceride-based systems mayinclude, for example, the following branded products (and correspondinggeneric products): Captex™ 355 EP (glyceryl tricaprylate/caprate, fromAbitec, Columbus Ohio), Crodamol™ GTC/C (medium chain triglyceride, fromCroda, Cowick Hall, UK) or Labrafac™ CC (medium chain triglyides, fromGattefosse), Captex™ 500P (glyceryl triacetate i.e. triacetin, fromAbitec), Capmul™ MCM (medium chain mono- and diglycerides, fromAbitec),Migyol™ 812 (caprylic/capric triglyceride, from Condea, Cranford N.J.),Migyol™ 829 (caprylic/capric/succinic triglyceride, from Condea),Migyol™ 840 (propylene glycol dicaprylate/dicaprate, from Condea),Labrafil™ M1944CS (oleoyl macrogol-6 glycerides, from Gattefosse),Peceol™ (glyceryl monooleate, from Gattefosse) and Maisine™ 35-1(glyceryl monooleate, from Gattefosse). Of particular interest are themedium chain (about C.sub.8 to C.sub.10) triglyceride oils. Thesesolvents frequently make up the predominant portion of the composition,i.e., greater than about 50%, usually greater than about 80%, forexample about 95% or 99%. Adjuvants and additives may also be includedwith the solvents principally as taste-mask agents, palatability andflavoring agents, antioxidants, stabilizers, texture and viscositymodifiers and solubilizers.

Suspensions, in addition to the compound of the present invention or thecombination, may further comprise carriers such as suspending agents,e.g., ethoxylated isostearyl alcohols, polyoxyethylene sorbitol andsorbitan esters, microcrystalline cellulose, aluminum metahydroxide,bentonite, agar-agar, and tragacanth, or mixtures of these substances,and the like.

Compositions for rectal or vaginal administration preferably comprisesuppositories, which can be prepared by mixing a compound of the presentinvention or a combination with suitable non-irritating excipients orcarriers, such as cocoa butter, polyethylene glycol or a suppository waxwhich are solid at ordinary room temperature, but liquid at bodytemperature, and therefore, melt in the rectum or vaginal cavity therebyreleasing the active component(s).

Dosage forms for topical administration of the compounds of the presentinvention or combinations include ointments, creams, lotions, powdersand sprays. The drugs are admixed with a pharmaceutically acceptableexcipient, diluent or carrier, and any preservatives, buffers, orpropellants that may be required.

Many of the present compounds are poorly soluble in water, e.g., lessthan about 1 μg/mL. Therefore, liquid compositions in solubilizing,non-aqueous solvents such as the medium chain triglyceride oilsdiscussed above are a preferred dosage form for these compounds.

Solid amorphous dispersions, including dispersions formed by aspray-drying process, are also a preferred dosage form for the poorlysoluble compounds of the invention. By “solid amorphous dispersion” ismeant a solid material in which at least a portion of the poorly solublecompound is in the amorphous form and dispersed in a water-solublepolymer. By “amorphous” is meant that the poorly soluble compound is notcrystalline. By “crystalline” is meant that the compound exhibitslong-range order in three dimensions of at least 100 repeat units ineach dimension. Thus, the term amorphous is intended to include not onlymaterial which has essentially no order, but also material which mayhave some small degree of order, but the order is in less than threedimensions and/or is only over short distances. Amorphous material maybe characterized by techniques known in the art such as powder X-raydiffraction (PXRD) crystallography, solid state NMR, or thermaltechniques such as differential scanning calorimetry (DSC).

Preferably, at least a major portion (i.e., at least about 60 wt %) ofthe poorly soluble compound in the solid amorphous dispersion isamorphous. The compound can exist within the solid amorphous dispersionin relatively pure amorphous domains or regions, as a solid solution ofthe compound homogeneously distributed throughout the polymer or anycombination of these states or those states that lie intermediatebetween them. Preferably, the solid amorphous dispersion issubstantially homogeneous so that the amorphous compound is dispersed ashomogeneously as possible throughout the polymer. As used herein,“substantially homogeneous” means that the fraction of the compound thatis present in relatively pure amorphous domains or regions within thesolid amorphous dispersion is relatively small, on the order of lessthan 20 wt %, and preferably less than 10 wt % of the total amount ofdrug.

Water-soluble polymers suitable for use in the solid amorphousdispersions should be inert, in the sense that they do not chemicallyreact with the poorly soluble compound in an adverse manner, arepharmaceutically acceptable, and have at least some solubility inaqueous solution at physiologically relevant pHs (e.g. 1-8). The polymercan be neutral or ionizable, and should have an aqueous-solubility of atleast 0.1 mg/mL over at least a portion of the pH range of 1-8.

Water-soluble polymers suitable for use with the present invention maybe cellulosic or non-cellulosic. The polymers may be neutral orionizable in aqueous solution. Of these, ionizable and cellulosicpolymers are preferred, with ionizable cellulosic polymers being morepreferred.

Exemplary water-soluble polymers include hydroxypropyl methyl celluloseacetate succinate (HPMCAS), hydroxypropyl methyl cellulose (HPMC),hydroxypropyl methyl cellulose phthalate (HPMCP), carboxy methyl ethylcellulose (CMEC), cellulose acetate phthalate (CAP), cellulose acetatetrimellitate (CAT), polyvinylpyrrolidone (PVP), hydroxypropyl cellulose(HPC), methyl cellulose (MC), block copolymers of ethylene oxide andpropylene oxide (PEO/PPO, also known as poloxamers), and mixturesthereof. Especially preferred polymers include HPMCAS, HPMC, HPMCP,CMEC, CAP, CAT, PVP, poloxamers, and mixtures thereof. Most preferred isHPMCAS. See European Patent Application Publication No. 0 901 786 A2,the disclosure of which is incorporated herein by reference.

The solid amorphous dispersions may be prepared according to any processfor forming solid amorphous dispersions that results in at least a majorportion (at least 60%) of the poorly soluble compound being in theamorphous state. Such processes include mechanical, thermal and solventprocesses. Exemplary mechanical processes include milling and extrusion;melt processes including high temperature fusion, solvent-modifiedfusion and melt-congeal processes; and solvent processes includingnon-solvent precipitation, spray coating and spray drying. See, forexample, the following U.S. patents, the pertinent disclosures of whichare incorporated herein by reference: U.S. Pat. Nos. 5,456,923 and5,939,099, which describe forming dispersions by extrusion processes;U.S. Pat. Nos. 5,340,591 and 4,673,564, which describe formingdispersions by milling processes; and U.S. Pat. Nos. 5,707,646 and4,894,235, which describe forming dispersions by melt congeal processes.In a preferred process, the solid amorphous dispersion is formed byspray drying, as disclosed in European Patent Application PublicationNo. 0 901 786 A2. In this process, the compound and polymer aredissolved in a solvent, such as acetone or methanol, and the solvent isthen rapidly removed from the solution by spray drying to form the solidamorphous dispersion. The solid amorphous dispersions may be prepared tocontain up to about 99 wt % of the compound, e.g., 1 wt %, 5 wt %, 10 wt%, 25 wt %, 50 wt %, 75 wt %, 95 wt %, or 98 wt % as desired.

The solid dispersion may be used as the dosage form itself or it mayserve as a manufacturing-use-product (MUP) in the preparation of otherdosage forms such as capsules, tablets, solutions or suspensions. Anexample of an aqueous suspension is an aqueous suspension of a 1:1 (w/w)compound/HPMCAS-HF spray-dried dispersion containing 2.5 mg/mL ofcompound in 2% polysorbate-80. Solid dispersions for use in a tablet orcapsule will generally be mixed with other excipients or adjuvantstypically found in such dosage forms. For example, an exemplary fillerfor capsules contains a 2:1 (w/w) compound/HPMCAS-MF spray-drieddispersion (60%), lactose (fast flow) (15%), microcrystalline cellulose(e.g., Avicel.sup.(R0-102) (15.8%), sodium starch (7%), sodium laurylsulfate (2%) and magnesium stearate (1%).

The HPMCAS polymers are available in low, medium and high grades asAqoa.sup.(R)-LF, Aqoat.sup.(R)-MF and Aqoat.sup.(R)—HF respectively fromShin-Etsu Chemical Co., LTD, Tokyo, Japan. The higher MF and HF gradesare generally preferred.

The following paragraphs describe exemplary formulations, dosages, etc.useful for non-human animals. The administration of the compounds of thepresent invention and combinations of the compounds of the presentinvention with anti-obesity agents can be effected orally or non-orally.

An amount of a compound of the present invention or combination of acompound of the present invention with another anti-obesity agent isadministered such that an effective dose is received. Generally, a dailydose that is administered orally to an animal is between about 0.01 andabout 1,000 mg/kg of body weight, e.g., between about 0.01 and about 300mg/kg or between about 0.01 and about 100 mg/kg or between about 0.01and about 50 mg/kg of body weight, or between about 0.01 and about 25mg/kg, or about 0.01 and about 10 mg/kg or about 0.01 and about 5 mg/kg.

Conveniently, a compound of the present invention (or combination) canbe carried in the drinking water so that a therapeutic dosage of thecompound is ingested with the daily water supply. The compound can bedirectly metered into drinking water, preferably in the form of aliquid, water-soluble concentrate (such as an aqueous solution of awater-soluble salt).

Conveniently, a compound of the present invention (or combination) canalso be added directly to the feed, as such, or in the form of an animalfeed supplement, also referred to as a premix or concentrate. A premixor concentrate of the compound in an excipient, diluent or carrier ismore commonly employed for the inclusion of the agent in the feed.Suitable excipients, diluents or carriers are liquid or solid, asdesired, such as water, various meals such as alfalfa meal, soybeanmeal, cottonseed oil meal, linseed oil meal, corncob meal and corn meal,molasses, urea, bone meal, and mineral mixes such as are commonlyemployed in poultry feeds. A particularly effective excipient, diluentor carrier is the respective animal feed itself; that is, a smallportion of such feed. The carrier facilitates uniform distribution ofthe compound in the finished feed with which the premix is blended.Preferably, the compound is thoroughly blended into the premix and,subsequently, the feed. In this respect, the compound may be dispersedor dissolved in a suitable oily vehicle such as soybean oil, corn oil,cottonseed oil, and the like, or in a volatile organic solvent and thenblended with the carrier. It will be appreciated that the proportions ofcompound in the concentrate are capable of wide variation since theamount of the compound in the finished feed may be adjusted by blendingthe appropriate proportion of premix with the feed to obtain a desiredlevel of compound.

High potency concentrates may be blended by the feed manufacturer withproteinaceous carrier such as soybean oil meal and other meals, asdescribed above, to produce concentrated supplements, which are suitablefor direct feeding to animals. In such instances, the animals arepermitted to consume the usual diet. Alternatively, such concentratedsupplements may be added directly to the feed to produce a nutritionallybalanced, finished feed containing a therapeutically effective level ofa compound of the present invention. The mixtures are thoroughly blendedby standard procedures, such as in a twin shell blender, to ensurehomogeneity.

If the supplement is used as a top dressing for the feed, it likewisehelps to ensure uniformity of distribution of the compound across thetop of the dressed feed.

Drinking water and feed effective for increasing lean meat depositionand for improving lean meat to fat ratio are generally prepared bymixing a compound of the present invention with a sufficient amount ofanimal feed to provide from about 0.001 to about 500 ppm of the compoundin the feed or water.

The preferred medicated swine, cattle, sheep and goat feed generallycontain from about 1 to about 400 grams of a compound of the presentinvention (or combination) per ton of feed, the optimum amount for theseanimals usually being about 50 to about 300 grams per ton of feed.

The preferred poultry and domestic pet feeds usually contain about 1 toabout 400 grams and preferably about 10 to about 400 grams of a compoundof the present invention (or combination) per ton of feed.

For parenteral administration in animals, the compounds of the presentinvention (or combination) may be prepared in the form of a paste or apellet and administered as an implant, usually under the skin of thehead or ear of the animal in which increase in lean meat deposition andimprovement in lean meat to fat ratio is sought.

Paste Formulations may be prepared by dispersing the drug in apharmaceutically acceptable oil such as peanut oil, sesame oil, corn oilor the like.

Pellets containing an effective amount of a compound of the presentinvention, pharmaceutical composition, or combination may be prepared byadmixing a compound of the present invention or combination with adiluent such as carbowax, carnuba wax, and the like, and a lubricant,such as magnesium or calcium stearate, may be added to improve thepelleting process.

It is, of course, recognized that more than one pellet may beadministered to an animal to achieve the desired dose level which willprovide the increase in lean meat deposition and improvement in leanmeat to fat ratio desired. Moreover, implants may also be madeperiodically during the animal treatment period in order to maintain theproper drug level in the animal's body.

Liposomes containing these agents and/or compounds of the invention areprepared by methods known in the art, such as described in U.S. Pat.Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation timeare disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomescan be generated by the reverse phase evaporation method with a lipidcomposition comprising phosphatidylcholine, cholesterol andPEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes areextruded through filters of defined pore size to yield liposomes withthe desired diameter.

These agents and/or the compounds of the invention may also be entrappedin microcapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington, The Science and Practice of Pharmacy, 20th Ed., MackPublishing (2000).

Sustained-release preparations may be used. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the compound of the invention, whichmatrices are in the form of shaped articles, e.g., films, ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),or ‘poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919),copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as those used in LUPRON DEPOT™ (injectable microspheres composed oflactic acid-glycolic acid copolymer and leuprolide acetate), sucroseacetate isobutyrate, and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for intravenous administration must besterile. This is readily accomplished by, for example, filtrationthrough sterile filtration membranes. Compounds of the invention aregenerally placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

Suitable emulsions may be prepared using commercially available fatemulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ andLipiphysan™. The active ingredient may be either dissolved in apre-mixed emulsion composition or alternatively it may be dissolved inan oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil,corn oil or almond oil) and an emulsion formed upon mixing with aphospholipid (e.g., egg phospholipids, soybean phospholipids or soybeanlecithin) and water. It will be appreciated that other ingredients maybe added, for example glycerol or glucose, to adjust the tonicity of theemulsion. Suitable emulsions will typically contain up to 20% oil, forexample, between 5 and 20%. The fat emulsion can comprise fat dropletsbetween 0.1 and 1.0 μm, particularly 0.1 and 0.5 μm, and have a pH inthe range of 5.5 to 8.0.

The emulsion compositions can be those prepared by mixing a compound ofthe invention with Intralipid™ or the components thereof (soybean oil,egg phospholipids, glycerol and water).

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as set outabove. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions in preferably sterile pharmaceutically acceptable solventsmay be nebulised by use of gases. Nebulised solutions may be breatheddirectly from the nebulising device or the nebulising device may beattached to a face mask, tent or intermittent positive pressurebreathing machine. Solution, suspension or powder compositions may beadministered, preferably orally or nasally, from devices which deliverthe formulation in an appropriate manner.

The compounds herein may be formulated for oral, buccal, intranasal,parenteral (e.g., intravenous, intramuscular or subcutaneous) or rectaladministration or in a form suitable for administration by inhalation.The compounds of the invention may also be formulated for sustaineddelivery.

Methods of preparing various pharmaceutical compositions with a certainamount of active ingredient are known, or will be apparent in light ofthis disclosure, to those skilled in this art. For examples of methodsof preparing pharmaceutical compositions see Remington's PharmaceuticalSciences, 20th Edition (Lippincott Williams & Wilkins, 2000).

Pharmaceutical compositions according to the invention may contain0.1%-95% of the compound(s) of this invention, preferably 1%-70%. In anyevent, the composition to be administered will contain a quantity of acompound(s) according to the invention in an amount effective to treatthe disease/condition of the subject being treated.

Since the present invention has an aspect that relates to the treatmentof the disease/conditions described herein with a combination of activeingredients which may be administered separately, the invention alsorelates to combining separate pharmaceutical compositions in kit form.The kit comprises two separate pharmaceutical compositions: a compoundof Formula I a prodrug thereof or a salt of such compound or prodrug anda second compound as described above. The kit comprises a means forcontaining the separate compositions such as a container, a dividedbottle or a divided foil packet. Typically the kit comprises directionsfor the administration of the separate components. The kit form isparticularly advantageous when the separate components are preferablyadministered in different dosage forms (e.g., oral and parenteral), areadministered at different dosage intervals, or when titration of theindividual components of the combination is desired by the prescribingphysician.

An example of such a kit is a so-called blister pack. Blister packs arewell known in the packaging industry and are being widely used for thepackaging of pharmaceutical unit dosage forms (tablets, capsules, andthe like). Blister packs generally consist of a sheet of relativelystiff material covered with a foil of a preferably transparent plasticmaterial. During the packaging process recesses are formed in theplastic foil. The recesses have the size and shape of the tablets orcapsules to be packed. Next, the tablets or capsules are placed in therecesses and the sheet of relatively stiff material is sealed againstthe plastic foil at the face of the foil which is opposite from thedirection in which the recesses were formed. As a result, the tablets orcapsules are sealed in the recesses between the plastic foil and thesheet. Preferably the strength of the sheet is such that the tablets orcapsules can be removed from the blister pack by manually applyingpressure on the recesses whereby an opening is formed in the sheet atthe place of the recess. The tablet or capsule can then be removed viasaid opening.

It may be desirable to provide a memory aid on the kit, e.g., in theform of numbers next to the tablets or capsules whereby the numberscorrespond with the days of the regimen which the tablets or capsules sospecified should be ingested. Another example of such a memory aid is acalendar printed on the card, e.g., as follows “First Week, Monday,Tuesday, etc. . . . Second Week, Monday, Tuesday, . . . ” etc. Othervariations of memory aids will be readily apparent. A “daily dose” canbe a single tablet or capsule or several pills or capsules to be takenon a given day. Also, a daily dose of Formula I compound can consist ofone tablet or capsule while a daily dose of the second compound canconsist of several tablets or capsules and vice versa. The memory aidshould reflect this.

In another specific embodiment of the invention, a dispenser designed todispense the daily doses one at a time in the order of their intendeduse is provided. Preferably, the dispenser is equipped with amemory-aid, so as to further facilitate compliance with the regimen. Anexample of such a memory-aid is a mechanical counter which indicates thenumber of daily doses that has been dispensed. Another example of such amemory-aid is a battery-powered micro-chip memory coupled with a liquidcrystal readout, or audible reminder signal which, for example, readsout the date that the last daily dose has been taken and/or reminds onewhen the next dose is to be taken.

Also, as the present invention has an aspect that relates to thetreatment of the disease/conditions described herein with a combinationof active ingredients which may be administered jointly, the inventionalso relates to combining separate pharmaceutical compositions in asingle dosage form, such as (but not limited to) a single tablet orcapsule, a bilayer or multilayer tablet or capsule, or through the useof segregated components or compartments within a tablet or capsule.

The active ingredient may be delivered as a solution in an aqueous ornon-aqueous vehicle, with or without additional solvents, co-solvents,excipients, or complexation agents selected from pharmaceuticallyacceptable diluents, excipients, vehicles, or carriers.

The active ingredient may be formulated as a solid dispersion or as aself emulsified drug delivery system (SEDDS) with pharmaceuticallyacceptable excipients.

The active ingredient may be formulated as an immediate release ormodified release tablet or capsule. Alternatively, the active ingredientmay be delivered as the active ingredient alone within a capsule shell,without additional excipients.

Experimental Procedures

The following illustrate the synthesis of various compounds of thepresent invention. Additional compounds within the scope of thisinvention may be prepared using the methods illustrated in theseExamples, either alone or in combination with techniques generally knownin the art.

Experiments were generally carried out under inert atmosphere (nitrogenor argon), particularly in cases where oxygen- or moisture-sensitivereagents or intermediates were employed. Commercial solvents andreagents were generally used without further purification. Anhydroussolvents were employed where appropriate, generally AcroSeal® productsfrom Acros Organics, Aldrich® Sure/Sear™ from SigmaAldrich, or DriSoly®products from EMD Chemicals. In other cases, commercial solvents werepassed through columns packed with 4 Å molecular sieves, until thefollowing QC standards for water were attained: a)<100 ppm fordichloromethane, toluene, N,N-dimethylformamide, and tetrahydrofuran;b)<180 ppm for methanol, ethanol, 1,4-dioxane, and diisopropylamine. Forvery sensitive reactions, solvents were further treated with metallicsodium, calcium hydride, or molecular sieves, and distilled just priorto use. Products were generally dried under vacuum before being carriedon to further reactions or submitted for biological testing. Massspectrometry data is reported from liquid chromatography-massspectrometry (LCMS), atmospheric pressure chemical ionization (APCI)instrumentation. Chemical shifts for nuclear magnetic resonance (NMR)data are expressed in parts per million (ppm, δ) referenced to residualpeaks from the deuterated solvents employed

Reactions proceeding through detectable intermediates were generallymonitored by LCMS, and allowed to proceed to full conversion prior toaddition of subsequent reagents. For syntheses referencing procedures inother Examples or Methods, reaction conditions (reaction time andtemperature) may vary. In general, reactions were monitored bythin-layer chromatography or mass spectrometry, and subjected to work-upwhen appropriate. Purifications may vary between experiments: ingeneral, solvents and the solvent ratios used for eluents/gradients werechosen to provide appropriate Rfs or retention times. All startingmaterials in these Preparations and Examples are either commerciallyavailable or can be prepared by methods known in the art or as describedherein.

Unless specified otherwise, starting materials are generally availablefrom commercial sources such as Aldrich Chemicals Co. (Milwaukee, Wis.),Lancaster Synthesis, Inc. (Windham, N.H.), Acros Organics (Fairlawn,N.J.), Maybridge Chemical Company, Ltd. (Cornwall, England) and TygerScientific (Princeton, N.J.). Certain common abbreviations and acronymshave been employed which may include: AcOH (acetic acid), DBU(1,8-diazabicyclo[5.4.0]undec-7-ene), CDI (1,1′-carbonyldiimidazole),DCM (dichloromethane), DEA (diethylamine), DIPEA(N,N-diisopropylethylamine), DMAP (4-dimethylaminopyridine), DMF(N,N-dimethylformamide), DMSO (dimethyl sulfoxide), EDCl[N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide], Et₂O (diethyl ether),EtOAc (ethyl acetate), EtOH (ethanol), HATU[2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium], HBTU(O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluoro phosphate),HOBT (1-hydroxybenzotriazole), IPA (2-propanol), KHMDS [potassiumbis(trimethylsilyl)amide], MeOH (methanol), MTBE (tert-butyl methylether), NaBH(OAc)₃ (sodium triacetoxyborohydride), NaHMDS [sodiumbis(trimethylsilyl)amide], NMP (N-methylpyrrolidone), SEM{[2-(trimethylsilyl)ethoxy]methyl}, TEA (triethylamine), TFA(trifluoroacetic acid), THF (tetrahydrofuran), and T₃P (propanephosphonic acid anhydride).

Reactions were performed in air or, when oxygen- or moisture-sensitivereagents or intermediates were employed, under an inert atmosphere(nitrogen or argon). When appropriate, reaction apparatuses were driedunder dynamic vacuum using a heat gun, and anhydrous solvents(Sure-Seal™ products from Aldrich Chemical Company, Milwaukee, Wis. orDriSolv™ products from EMD Chemicals, Gibbstown, N.J.) were employed.Commercial solvents and reagents were used without further purification.When indicated, reactions were heated by microwave irradiation usingBiotage Initiator or Personal Chemistry Emrys Optimizer microwaves orthe like. Reaction progress was monitored using thin-layerchromatography (TLC), liquid chromatography-mass spectrometry (LCMS) andhigh-performance liquid chromatography (HPLC) analyses. TLC wasperformed on pre-coated silica gel plates with a fluorescence indicator(254 nm excitation wavelength) and visualized under UV light and/or withI₂, KMnO₄, CoCl₂, phosphomolybdic acid, and/or ceric ammonium molybdatestains. LCMS data were acquired on an Agilent 1100 Series instrumentwith a Leap Technologies autosampler, Gemini C₁₈ columns,acetonitrile/water gradients, and either trifluoroacetic acid, formicacid, or ammonium hydroxide modifiers or similar equipment. The columneluent was analyzed using a Waters ZQ mass spectrometer scanning in bothpositive and negative ion modes from 100 to 1200 Da. Other similarinstruments were also used. HPLC data were acquired on an Agilent 1100Series instrument using Gemini or XBridge C18 columns,acetonitrile/water gradients, and either trifluoroacetic acid orammonium hydroxide modifiers and comparable equipment. Purificationswere performed by medium performance liquid chromatography (MPLC) usingIsco CombiFlash Companion, AnaLogix IntelliFlash 280, Biotage SP1, orBiotage Isolera One instruments and pre-packed Isco RediSep or BiotageSnap silica cartridges and the like. Chiral purifications were performedby chiral supercritical fluid chromatography (SFC) using Berger or Tharinstruments and similar instruments; ChiralPAK-AD, -AS, —IC,Chiralcel-OD, or -OJ columns; and CO₂ mixtures with MeOH, EtOH, iPrOH,or acetonitrile, alone or modified using trifluoroacetic acid or iPrNH₂.UV detection was used to trigger fraction collection.

Mass spectrometry data are reported from LCMS analyses. Massspectrometry (MS) was performed via atmospheric pressure chemicalionization (APCI), electrospray Ionization (ESI), electron impactionization (EI) or electron scatter (ES) ionization sources. Protonnuclear magnetic spectroscopy (¹H NMR) chemical shifts are given inparts per million downfield from tetramethylsilane and were recorded onon 300, 400, 500, or 600 MHz Varian spectrometers. Chemical shifts areexpressed in parts per million (ppm, δ) referenced to the deuteratedsolvent residual peaks. The peak shapes are described as follows: s,singlet; d, doublet; t, triplet; q, quartet; quin, quintet; m,multiplet; br s, broad singlet; app, apparent. Analytical SFC data wereacquired on a Berger analytical instrument as described above. Opticalrotation data were acquired on a PerkinElmer model 343 polarimeter usinga 1 dm cell. Silica gel chromatography was performed primarily using amedium pressure Biotage or ISCO systems using columns pre-packaged byvarious commercial vendors including Biotage and ISCO. Unless otherwisenoted, chemical reactions were performed at room temperature (about 23degrees Celsius).

The compounds and intermediates described below were named using thenaming convention provided with ACD/ChemSketch 2017.2.1, File VersionN40E41, Build 96719 (Advanced Chemistry Development, Inc., Toronto,Ontario, Canada). The naming convention provided with ACD/ChemSketch2017.2.1 is well known by those skilled in the art and it is believedthat the naming convention provided with ACD/ChemSketch 2017.2.1generally comports with the IUPAC (International Union for Pure andApplied Chemistry) recommendations on Nomenclature of Organic Chemistryand the CAS Index rules.

The terms “concentrated”, “evaporated”, and “concentrated in vacuo”refer to the removal of solvent at reduced pressure on a rotaryevaporator with a bath temperature less than 60° C. The abbreviation“min” and “h” stand for “minutes” and “hours” respectively. “Roomtemperature” or “ambient temperature” means a temperature between 15° C.and 25° C., and “UPLC” refers to ultra-performance liquidchromatography,

Hydrogenation may be performed in a Parr shaker under pressurizedhydrogen gas, or in a Thales-nano H-Cube flow hydrogenation apparatus atfull hydrogen and a flow rate between 1-2 mL/min at specifiedtemperature.

HPLC, UPLC, LCMS, and SFC retention times were measured using themethods noted in the procedures.

Example 1

Step 1. Synthesis of 2-(thieno[3,2-b]thiophen-2-yl)-1,3-dioxolane (C1)

This reaction was carried out in three parallel batches. Pyridiniump-toluenesulfonate (11.2 g, 44.6 mmol) was added to a solution ofethylene glycol (133 mL, 2.38 mol) andthieno[3,2-b]thiophene-2-carbaldehyde (100 g, 594 mmol) in toluene (1L), and the reaction mixture was heated at 125° C. for 16 hours, in anapparatus equipped with a Dean-Stark trap. The resulting mixture waswashed sequentially with water (3×1 L) and saturated aqueous sodiumcarbonate solution (1 L), and the organic layer was dried over sodiumsulfate, filtered, and concentrated in vacuo. Silica gel chromatography(Gradient: 1% to 25% ethyl acetate in petroleum ether) afforded C1 as awhite solid. Combined yield: 264 g, 1.24 mol, 70%. GCMS m/z 212 [M⁺]. ¹HNMR (400 MHz, chloroform-d) δ 7.38 (d, J=5.3 Hz, 1H), 7.35 (br s, 1H),7.24 (dd, J=5.3, 0.7 Hz, 1H), 6.17 (s, 1H), 4.20-4.00 (m, 4H).

Step 2. Synthesis of[5-(1,3-dioxolan-2-yl)-2-fluorothieno[3,2-b]thiophen-3-yl](trimethyl)silane(C2)

A solution of C1 (40.0 g, 188 mmol) in a mixture of toluene (920 mL) andtetrahydrofuran (720 mL) was cooled to −78° C. n-Butyllithium (2.5 Msolution in hexanes; 82.9 mL, 207 mmol) was added in a drop-wise manner,at a rate such that the internal temperature of the reaction mixtureremained below −72° C. After the addition had been completed, thereaction mixture was stirred at −78° C. for 2 hours, whereupon asolution of N-fluoro-N-(phenylsulfonyl)benzenesulfonamide (71.3 g, 226mmol) in tetrahydrofuran (200 mL) was added in a drop-wise manner usingan addition funnel, at a rate that maintained the internal reactiontemperature below −72° C. The reaction mixture was allowed to stir at−78° C. for 1 hour, and then quenched at −78° C. by addition ofsaturated aqueous sodium bicarbonate solution (1.5 L). After theresulting mixture had warmed to room temperature, the organic layer waswashed with aqueous sodium bicarbonate solution (3×500 mL), and thecombined aqueous layers were extracted with ethyl acetate (500 mL). Theethyl acetate extract was washed with aqueous sodium bicarbonatesolution (3×200 mL) and then combined with the first organic layer,dried over sodium sulfate, filtered, and concentrated in vacuo. Theresulting solid was triturated with a mixture of heptane and diethylether (1:1; 250 mL), and the supernatant was decanted, leaving a stickysolid. This solid was again triturated with a mixture of heptane anddiethyl ether (1:1; 2×100 mL), and the supernatant was decanted; thecombined decanted solutions were concentrated under reduced pressure toprovide a greenish solid (39 g).

This solid (39 g) was dissolved in a mixture of toluene (760 mL) andtetrahydrofuran (608 mL), cooled to −78° C., and treated in a drop-wisemanner with n-butyllithium (2.5 M solution in hexanes; 98.0 mL, 245mmol) at a rate that maintained the internal reaction temperature below−72° C. After the reaction mixture had been stirred at −78° C. for 1hour, a solution of trimethylsilyl chloride (31.1 mL, 245 mmol) intetrahydrofuran (35 mL) was added, while again maintaining the internalreaction temperature below −72° C. The reaction mixture was then stirredat −78° C. for 45 minutes, whereupon an aliquot was partitioned betweendiethyl ether and saturated aqueous sodium bicarbonate solution. GCMSanalysis of the organic layer of this aliquot indicated conversion toC2: GCMS m/z 302.1 [M⁺]. The reaction mixture was quenched at −78° C. byaddition of saturated aqueous sodium bicarbonate solution (1.5 L), andthe resulting mixture was allowed to warm to room temperature. Theorganic layer was washed with aqueous sodium bicarbonate solution (3×500mL), and the combined aqueous layers were extracted with ethyl acetate(500 mL). The ethyl acetate extract was washed with aqueous sodiumbicarbonate solution (3×200 mL) and then combined with the first organiclayer, dried over sodium sulfate, filtered, and concentrated in vacuo.The residue was dissolved in dichloromethane (30 mL) and treated withsilica gel (180 g); the resulting slurry was loaded on top of a plug ofdiatomaceous earth and eluted with dichloromethane (4 L) until C2 ceasedto elute. Concentration of these fractions under reduced pressureprovided an oily, orange residue (49 g), which was purified via silicagel chromatography (Eluent: 30% dichloromethane in heptane) to afford C2as a yellow solid. Yield: 36.0 g, 119 mmol, 63%. ¹H NMR (400 MHz,methanol-d₄) δ 7.34 (s, 1H), 6.10 (s, 1H), 4.15-3.96 (m, 4H), 0.39 (s,9H).

Step 3. Synthesis of2-(3-chloro-5-fluorothieno[3,2-b]thiophen-2-yl)-1,3-dioxolane (C4)

Lithium diisopropylamide (2.0 M solution intetrahydrofuran/heptane/ethylbenzene; 90.3 mL, 181 mmol) was added in adrop-wise manner to a −78° C. solution of C2 (36.4 g, 120 mmol) intetrahydrofuran (1.2 L), at a rate that maintained the internaltemperature below −72° C. After completion of the addition, the reactionmixture was allowed to stir at −78° C. for 3 hours, whereupon a solutionof hexachloroethane (37.0 g, 156 mmol) in tetrahydrofuran (60 mL) wasadded drop-wise, in a manner that maintained the internal reactiontemperature below −72° C. The reaction mixture was stirred for a further30 minutes at −78° C., at which time the cooling bath was removed, andthe reaction mixture was allowed to warm to room temperature overnight.An aliquot of the reaction mixture was partitioned between diethyl etherand saturated aqueous sodium bicarbonate solution; GCMS analysis of theorganic layer indicated conversion to intermediate C3{[6-chloro-5-(1,3-dioxolan-2-yl)-2-fluorothieno[3,2-b]thiophen-3-yl](trimethyl)silane}:GCMS m/z 336.1 [M⁺]. The reaction mixture was diluted with saturatedaqueous sodium bicarbonate solution and extracted three times with ethylacetate. The combined organic extracts were washed with saturatedaqueous sodium chloride solution, dried over sodium sulfate, filtered,and concentrated in vacuo to provide C3 as a dark amber oil (40.5 g).This material was dissolved in tetrahydrofuran (1.13 L) and treated withwater (7.36 mL, 408 mmol), followed by tetrabutylammonium fluoride (1 Msolution in tetrahydrofuran; 180 mL, 180 mmol). After the reactionmixture had been stirred at room temperature for 15 minutes, GCMSanalysis indicated conversion to C4: GCMS m/z 264.0 [M⁺]. The reactionmixture was poured into saturated aqueous sodium bicarbonate solution,and extracted three times with ethyl acetate; the combined organiclayers were washed with saturated aqueous sodium chloride solution,dried over sodium sulfate, filtered, and concentrated under reducedpressure. Silica gel chromatography (Gradient: 0% to 5% ethyl acetate inheptane) provided C4 as an oily, orange residue. Yield: 17.2 g, 65.0mmol, 54%. ¹H NMR (400 MHz, methanol-d₄) δ 7.02 (d, J=1.6 Hz, 1H), 6.19(s, 1H), 4.17-3.97 (m, 4H).

Step 4. Synthesis of3-chloro-5-fluorothieno[3,2-b]thiophene-2-carbaldehyde (C5)

A solution of hydrogen chloride in 1,4-dioxane (4.0 M; 163 mL, 652 mmol)was added to a solution of C4 (17.2 g, 65.0 mmol) in a mixture of1,4-dioxane (575 mL) and water (57.5 mL). The reaction mixture wasstirred at room temperature for 1 hour, whereupon it was partitionedbetween ethyl acetate (200 mL) and water (500 mL). The organic layer waswashed with saturated aqueous sodium chloride solution (500 mL). Thesaturated aqueous sodium chloride layer was combined with the originalaqueous layer and extracted with ethyl acetate (2×200 mL); theseextracts were combined with the first organic layer, dried over sodiumsulfate, filtered, and concentrated in vacuo to provide a brown solid.This solid was mixed with pentane (100 mL) and stirred vigorously for 20minutes at room temperature. The resulting solid was collected viafiltration and washed with pentane (3×20 mL), affording C5 as anoff-white solid. Yield: 13.0 g, 58.9 mmol, 91%. GCMS m/z 191.0 [M-CHO]⁺.¹H NMR (400 MHz, chloroform-d) δ 10.07 (s, 1H), 6.87 (d, J=1.2 Hz, 1H).

Step 5. Synthesis of3-chloro-5-fluorothieno[3,2-b]thiophene-2-carboxylic acid (1)

A solution of sodium chlorite (20.5 g, 227 mmol) and sodium dihydrogenphosphate (27.5 g, 229 mmol) in water (100 mL) was added slowly, in adrop-wise manner, to a 0° C. solution of C5 (10.0 g, 45.3 mmol) in amixture of dimethyl sulfoxide (56 mL) and 2-methyltetrahydrofuran (100mL). The reaction mixture was then allowed to warm to room temperature,and was stirred at that temperature until the starting material had beencompletely consumed, as assessed by LCMS analysis (approximately 2hours). The reaction mixture was then poured in portions into a cold (0°C.) saturated aqueous solution of sodium thiosulfate pentahydrate (300mL), at a rate that maintained the temperature of the resulting mixturebelow 15° C. After stirring at 10° C. for 20 minutes, the mixture wasdiluted with ethyl acetate (200 mL). The aqueous layer was extractedwith ethyl acetate (2×200 mL), and the combined organic layers werewashed with saturated aqueous sodium chloride solution, dried overmagnesium sulfate, filtered, and concentrated in vacuo. The residue wasstirred in a mixture of heptane and ethyl acetate (9:1, 50 mL) for about1 hour. The resulting solid was collected via filtration and washed witha mixture of heptane and ethyl acetate (9:1, 2×20 mL), providing a whitesolid (10.58 g). This was stirred in dichloromethane for 20 minutes andfiltered; the filter cake was washed with dichloromethane (2×20 mL) toafford 3-chloro-5-fluorothieno[3,2-b]thiophene-2-carboxylic acid as awhite solid. Yield: 10.0 g, 42.2 mmol, 93%. LCMS m/z 191.0 (chlorineisotope pattern observed) [(M-CO₂)—H]⁻. ¹H NMR (400 MHz, DMSO-d₆) δ13.5(br s, 1H), 7.41 (d, J=1.7 Hz, 1H).

Example 2

Step 1. Synthesis of[6-bromo-5-(1,3-dioxolan-2-yl)-2-fluorothieno[3,2-b]thiophen-3-yl](trimethyl)silane(C6)

Lithium diisopropylamide solution (2 M; 9.31 mL, 18.6 mmol) was added ina drop-wise manner to a −65° C. solution of C2 (4.33 g, 14.3 mmol) intetrahydrofuran (140 mL), at a rate that maintained the internalreaction temperature below −60° C. After completion of the addition, thereaction mixture was stirred at −60° C. for 3 hours, whereuponN-bromosuccinimide (3.82 g, 21.5 mmol) was added, and the reactionmixture was allowed to warm to 18° C. and stir for 16 hours. It was thencombined with a similar reaction carried out using C2 (3.00 g, 9.92mmol), and partitioned between aqueous sodium bicarbonate solution (300mL) and ethyl acetate. The aqueous layer was extracted with ethylacetate (3×200 mL); the combined organic layers were washed withsaturated aqueous sodium chloride solution (400 mL), dried over sodiumsulfate, filtered, and concentrated in vacuo, affording a white-brownsolid (9.24 g), which was used directly in the following step. By ¹H NMRanalysis, this material was an approximately 1:1 mixture of C6 and C2.¹H NMR (400 MHz, chloroform-d), C6 peaks only: δ 6.18 (s, 1H), 4.21-4.1(m, 2H), 4.08-4.00 (m, 2H), 0.37 (d, J=0.9 Hz, 9H).

Step 2. Synthesis of2-(3-bromo-5-fluorothieno[3,2-b]thiophen-2-yl)-1,3-dioxolane (C7)

To a solution of C6 and C2 (from the previous step; 9.24 g, <24.2 mmol)in tetrahydrofuran (120 mL) and water (0.437 mL, 24.2 mmol) was addedtetrabutylammonium fluoride (1 M solution in tetrahydrofuran; 36.4 mL,36.4 mmol). After the reaction mixture had been stirred at 20° C. for 1hour, it was poured into saturated aqueous sodium bicarbonate solution(70 mL) at 0° C., and the resulting mixture was stirred, then extractedwith ethyl acetate (3×60 mL). The combined organic layers were washedwith saturated aqueous sodium chloride solution, dried over sodiumsulfate, filtered, and concentrated in vacuo to provide C7 (7.49 g) as abrown solid. This material was used directly in the following step.

Step 3. Synthesis of3-bromo-5-fluorothieno[3,2-b]thiophene-2-carbaldehyde (C8)

To a solution of C7 (from the previous step; 7.49 g, <24.2 mmol) intetrahydrofuran (120 mL) was added hydrochloric acid (2 M; 12 mL) in adrop-wise manner. The reaction mixture was stirred at 40° C. for 2hours, whereupon it was diluted with water (100 mL) and extracted withethyl acetate (3×80 mL). The combined organic layers were washed withsaturated aqueous sodium chloride solution, dried over sodium sulfate,filtered, concentrated in vacuo, and purified via silica gelchromatography (Gradient: 0% to 20% ethyl acetate in petroleum ether) toafford C8 as a white solid. Yield: 1.35 g, 5.09 mmol, 21% over threesteps. ¹H NMR (400 MHz, chloroform-d) δ 9.99 (s, 1H), 6.91 (d, J=1.2 Hz,1H).

Step 4. Synthesis of 3-bromo-5-fluorothieno[3,2-b]thiophene-2-carboxylicacid (2)

A solution of C8 (1.35 g, 5.09 mol) in acetonitrile (20 mL) was cooledto 0° C. A solution of sodium dihydrogen phosphate (794 mg, 6.62 mmol)in water (1 mL) and an aqueous solution of hydrogen peroxide (30%; 2.6mL, 25 mmol) were added, followed by addition of a solution of sodiumchlorite (599 mg, 6.62 mmol) in water (3 mL) over 5 minutes. Theresulting biphasic reaction mixture was vigorously stirred at 0° C. for2 hours, then at 18° C. for 16 hours, whereupon it was poured intoaqueous sodium sulfite solution (20 mL). The resulting mixture wasstirred for 10 minutes, and then the pH of the mixture was adjusted toapproximately 1. The mixture was extracted with ethyl acetate (3×40 mL),and the combined organic layers were washed with saturated aqueoussodium chloride solution (50 mL), dried over sodium sulfate, filtrated,and concentrated in vacuo. The residue was stirred in petroleum ether(10 mL) for 30 minutes, and then filtered, providing3-bromo-5-fluorothieno[3,2-b]thiophene-2-carboxylic acid as a whitesolid. Yield: 964 mg, 3.43 mmol, 67%. LCMS m/z 234.9 (bromine isotopepattern observed) [(M-CO₂)+H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 13.7-12.9(br s, 1H), 7.45 (d, J=1.8 Hz, 1H).

Example 3

Step 1. Synthesis of 1-(3-bromo-2-thiophenyl)-2,2-difluoroethanone (C9)

Lithium diisopropylamide (2.0 M; 73.6 mL, 147 mmol) was added drop-wiseto a −70° C. solution of 3-bromothiophene (20.0 g, 123 mmol) intetrahydrofuran (240 mL). After the reaction mixture had been stirred at−70° C. for 1 hour, ethyl difluoroacetate (14.8 mL, 141 mmol) was addeddrop-wise at −78° C. Stirring was continued at that temperature for onehour, whereupon the reaction mixture was slowly warmed to 15° C. andstirred at 15° C. overnight. Hydrochloric acid (1 M; 500 mL) was added,followed by ethyl acetate (500 mL); the organic layer was washed withsaturated aqueous sodium chloride solution (500 mL) and filtered, andthe filtrate was concentrated in vacuo to afford C9 (29.6 g) as a brownoil. This material was used directly in the following step. ¹H NMR (400MHz, methanol-d₄) δ 7.51-7.45 (m, 1H), 7.04-6.99 (m, 1H), 6.07 (t,J_(HF)=55.7 Hz, 1H).

Step 2. Synthesis of ethyl3-(difluoromethyl)thieno[3,2-b]thiophene-2-carboxylate (C10)

Ethyl mercaptoacetate (13.4 mL, 122 mmol) was added in a drop-wisemanner to a 65° C. mixture of C9 (from the previous step; 29.6 g, 123mmol) and potassium carbonate (76.3 g, 552 mmol) inN,N-dimethylformamide. After the reaction mixture had been stirred at65° C. for 16 hours, it was poured into water (400 mL) and extractedwith ethyl acetate (2×400 mL). The combined organic layers were washedsequentially with aqueous lithium chloride solution (3%; 3×400 mL) andsaturated aqueous sodium chloride solution (2×400 mL), dried over sodiumsulfate, filtered, and concentrated in vacuo. Chromatography on silicagel (Gradient: 0% to 10% ethyl acetate in petroleum ether) provided C10as a yellow solid. Yield: 22.6 g, 86.2 mmol, 70% over 2 steps. ¹H NMR(400 MHz, chloroform-d) δ 7.67 (d, J=5.3 Hz, 1H), 7.56 (t, J_(HF)=55.1Hz, 1H), 7.29 (br d, J=5.4 Hz, 1H), 4.41 (q, J=7.1 Hz, 2H), 1.42 (t,J=7.1 Hz, 3H).

Step 3. Synthesis of[3-(difluoromethyl)thieno[3,2-b]thiophen-2-yl]methanol (C11)

A mixture of lithium aluminum hydride (6.40 g, 169 mmol) intetrahydrofuran (500 mL) was added drop-wise to a 0° C. solution of C10(22.1 g, 84.3 mmol) in tetrahydrofuran (300 mL), and the reactionmixture was stirred for 30 minutes at 0° C., then at 20° C. for 2 hours.it was then cooled to 0° C. and treated sequentially with water (6.4mL), aqueous sodium hydroxide solution (15%; 6.4 mL), and water (3×6.4mL). After filtration of the resulting mixture, the filter cake wasstirred with ethyl acetate (3×40 mL) for 10 minutes, and filtered; thecombined filtrates were concentrated in vacuo to afford C11 as a yellowsolid. This material was taken directly to the following step. ¹H NMR(400 MHz, chloroform-d) δ 7.43 (d, J=5.3 Hz, 1H), 7.24 (d, J=5.3 Hz,1H), 6.98 (t, J_(HF)=55.6 Hz, 1H), 4.95 (br s, 2H), 2.10 (br s, 1H).

Step 4. Synthesis of3-(difluoromethyl)thieno[3,2-b]thiophene-2-carbaldehyde (C12)

To a solution of C11 (from the previous step; 84.3 mmol) indichloromethane (420 mL) was added manganese(IV) oxide (73.3 g, 843mmol). After the reaction mixture had been stirred at 20° C. for 16hours, it was filtered; the filtrate was concentrated in vacuo toprovide C12 as a yellow solid. Yield: 14.0 g, 64.1 mmol, 76% over 2steps. ¹H NMR (400 MHz, chloroform-d) δ 10.08 (s, 1H), 7.76 (d, J=5.3Hz, 1H), 7.36 (d, J=5.3 Hz, 1H), 7.34 (t, J_(HF)=55.0 Hz, 1H).

Step 5. Synthesis of2-[3-(difluoromethyl)thieno[3,2-b]thiophen-2-yl]-1,3-dioxolane (C13)

1,2-Ethanediol (17.8 mL, 319 mmol) and p-toluenesulfonic acidmonohydrate (122 mg, 0.641 mmol) were added to a solution of C12 (14.0g, 64.1 mmol) in toluene (100 mL), and the reaction mixture was heatedat reflux (130° C.) overnight, while water generated from the reactionwas removed using a Dean-Stark trap. After the reaction mixture hadcooled to 15° C., saturated aqueous sodium bicarbonate solution (200 mL)was added, and the mixture was extracted with ethyl acetate (2×200 mL).The combined organic layers were washed sequentially with saturatedaqueous sodium bicarbonate solution (2×200 mL), water (2×200 mL), andsaturated aqueous sodium chloride solution (2×200 mL), then dried oversodium sulfate, filtered, and concentrated in vacuo to provide a yellowoil (16.0 g). A portion of this oil (15 g) was subjected to purificationvia silica gel chromatography (Gradient: 0% to 20% ethyl acetate inpetroleum ether) to afford C13 (14.3 g, 54.5 mmol) as a yellow oil.Adjusted yield: 15.2 g, 58.0 mmol, 90%. LCMS m/z 263.0 [M+H]⁺. ¹H NMR(400 MHz, chloroform-d) δ 7.45 (d, J=5.3 Hz, 1H), 7.24 (d, J=5.3 Hz,1H), 7.09 (t, J_(HF)=55.4 Hz, 1H), 6.35-6.32 (m, 1H), 4.17-4.03 (m, 4H).

Step 6. Synthesis of2-[3-(difluoromethyl)-5-fluorothieno[3,2-b]thiophen-2-yl]-1,3-dioxolane(C14)

n-Butyllithium (2.5 M solution; 3.66 mL, 9.15 mmol) was added in adrop-wise manner to a −70° C. solution of C13 (2.00 g, 7.62 mmol) intetrahydrofuran (30 mL). After the reaction mixture had been stirred at−70° C. for 4 hours, a solution ofN-fluoro-N-(phenylsulfonyl)benzenesulfonamide (2.89 g, 9.16 mmol) intetrahydrofuran (10 mL) was added, and the reaction mixture was warmedto 15° C. and stirred at 15° C. for 16 hours. Saturated aqueous sodiumbicarbonate solution (50 mL) was added, and the resulting mixture wasextracted with ethyl acetate (3×50 mL); the combined organic layers werewashed with saturated aqueous sodium chloride solution (100 mL), driedover sodium sulfate, filtered, and concentrated in vacuo to provide C14,which was progressed directly to the following step.

Step 7. Synthesis of3-(difluoromethyl)-5-fluorothieno[3,2-b]thiophene-2-carbaldehyde (C15)

To a solution of C14 (from the previous step; 7.62 mmol) intetrahydrofuran (20 mL) was added hydrochloric acid (2 M; 3.82 mL, 7.64mmol), and the reaction mixture was stirred at 40° C. for 2 hours. Itwas then concentrated in vacuo to remove tetrahydrofuran, and dilutedwith water (50 mL). The resulting mixture was extracted with ethylacetate (3×50 mL), and the combined organic layers were washed withsaturated aqueous sodium chloride solution (100 mL), dried over sodiumsulfate, filtered, concentrated in vacuo, and purified via silica gelchromatography (Gradient: 0% to 20% ethyl acetate in petroleum ether) toprovide a yellow solid (756 mg). By ¹H NMR analysis, this material wasan equimolar mixture of C15 and C12, which was progressed directly tothe following step. ¹H NMR (400 MHz, chloroform-d), peaks for C15 only:δ 10.01 (s, 1H), 7.31 (t, J_(HF)=55.0 Hz, 1H), 6.91-6.89 (m, 1H).

Step 8. Synthesis of3-(difluoromethyl)-5-fluorothieno[3,2-b]thiophene-2-carboxylic acid (3)

A solution of C15 (from the previous step, containing C12 as well; 756mg) and C15 [461 mg; derived from C13 (2.0 g, 7.6 mmol) through similarchemistry] in acetonitrile (20 mL) was cooled to 0° C. After a solutionof sodium dihydrogen phosphate (86.5 mg, 0.721 mmol) in water (1 mL) andan aqueous solution of hydrogen peroxide (30%; 2.63 mL, 23 mmol) hadbeen added, a solution of sodium chlorite (303 mg, 3.35 mmol) in water(3 mL) was added over 5 minutes. The resulting two-phase reactionmixture was vigorously stirred for 2 hours at 0° C., and then at roomtemperature (15° C.) for 16 hours. It was subsequently cooled to 0° C.,treated with sodium chlorite (303 mg, 3.35 mmol) and sodium dihydrogenphosphate (618 mg, 5.15 mmol), and stirred at 15° C. overnight. Thereaction mixture was then cooled to 10° C., quenched via addition of anaqueous solution of sodium sulfite (20 mL), and poured into an aqueoussolution of sodium sulfite (100 mL). Water (20 mL) was added, and theresulting mixture was adjusted to pH 1 by addition of 5 M hydrochloricacid; this provided a suspension, which was filtered. The collectedsolid was washed with water and purified via reversed-phase HPLC(Column: YMC-Actus Triart C18, 5 μm; Mobile phase A: water containing0.225% formic acid; Mobile phase B: acetonitrile; Gradient: 48% to 68%B) to provide3-(difluoromethyl)-5-fluorothieno[3,2-b]thiophene-2-carboxylic acid as awhite solid. Combined yield: 439 mg, 1.74 mmol, 11% over 3 steps. LCMSm/z 251.0 [M−H]⁻. ¹H NMR (400 MHz, DMSO-d₆) δ14.3-13.9 (br s, 1H), 7.63(t, J_(HF)=54.9 Hz, 1H), 7.43 (br s, 1H).

Example 4

Step 1. Synthesis of 5,6-difluorothieno[3,2-b]thiophene-2-carbaldehyde(C17)

To a −78° C. solution of C1 (4.05 g, 19.1 mmol) in tetrahydrofuran (191mL) was added n-butyllithium (2.5 M in hexanes, 9.92 mL, 24.8 mmol), ina drop-wise manner. After the reaction mixture had been stirred at −78°C. for 2 hours, a solution ofN-fluoro-N-(phenylsulfonyl)benzenesulfonamide (7.82 g, 24.8 mmol) intetrahydrofuran (20 mL) was added drop-wise. Stirring was continued at−78° C. for 30 minutes, whereupon the reaction mixture was allowed toslowly warm to room temperature. After 3 hours, it was cooled to −78° C.and treated drop-wise with n-butyllithium (2.5 M in hexanes; 11.4 mL,28.5 mmol), then stirred at −78° C. for 1 hour. A solution ofN-fluoro-N(phenylsulfonyl)benzenesulfonamide (10.2 g, 32.3 mmol) intetrahydrofuran (30 mL) was again added drop-wise, and the reactionmixture was allowed to warm to room temperature and stir overnight. GCMSanalysis at this point indicated a mixture of C16{2-(5,6-difluorothieno[3,2-b]thiophen-2-yl)-1,3-dioxolane, GCMS m/z248.0 [M]⁺} and its mono-fluoro analogue, presumed to be2-(5-fluorothieno[3,2-b]thiophen-2-yl)-1,3-dioxolane (GCMS m/z 230.0[M]⁺). Water (19 mL) was added, followed by a solution of hydrogenchloride in 1,4-dioxane (4.0 M; 28.6 mL, 114 mmol); stirring wascontinued at room temperature for 30 minutes, whereupon the reactionmixture was diluted with water and extracted with ethyl acetate. Thecombined organic layers were dried over sodium sulfate, filtered,concentrated in vacuo, and purified using silica gel chromatography(Gradient: 0% to 100% ethyl acetate in heptane) to afford C17 as anorange oil (3.06 g). This material was used directly in the followingstep. LCMS m/z 204.8 [M+H]⁺. LCMS also indicated the presence of amono-fluoro analogue, presumed to be5-fluorothieno[3,2-b]thiophene-2-carbaldehyde: LCMS m/z 186.9 [M+H]⁺.

Step 2. Synthesis of 5,6-difluorothieno[3,2-b]thiophene-2-carboxylicacid (4)

A solution of C17 (from the previous step, 3.06 g) in tetrahydrofuran(100 mL), water (25 mL), and 2-methyl-2-butene (25 mL) was treatedsequentially with sodium dihydrogen phosphate (8.99 g, 74.9 mmol) andsodium chlorite (6.78 g, 75.0 mmol). After the reaction mixture had beenstirred at room temperature for 1 hour, it was diluted with water andextracted with ethyl acetate. The combined organic layers were driedover sodium sulfate, filtered, concentrated in vacuo, and subjected tosilica gel chromatography (Gradient: 0% to 20% methanol indichloromethane), then purified via reversed-phase chromatography on aC18 column [Gradient: 25% to 100% water (containing 0.01%trifluoroacetic acid) in acetonitrile (containing 0.01% trifluoroaceticacid)]. The resulting solid was stirred in water, filtered, and washedwith water, then dissolved in ethanol and filtered; concentration of thefiltrate in vacuo provided a solid, which was then precipitated from anethanol solution upon cooling to provide5,6-difluorothieno[3,2-b]thiophene-2-carboxylic acid as a white solid.Yield: 1.19 g, 5.40 mmol, 28% over 2 steps. LCMS m/z 219.1 [M−H]⁻. ¹HNMR (400 MHz, DMSO-d₆) δ 13.6-13.5 (br s, 1H), 8.12 (d, J=2.1 Hz, 1H).

Example 5

Step 1. Synthesis of3,5-difluoro-6-(trimethylsilyl)thieno[3,2-b]thiophene-2-carbaldehyde(C19)

Lithium diisopropylamide (2.0 M solution intetrahydrofuran/heptane/ethylbenzene; 15.4 mL, 30.8 mmol) was added in adrop-wise manner to a −78° C. solution of C2 (6.22 g, 20.6 mmol) intetrahydrofuran (137 mL). After the reaction mixture had been stirred at−78° C. for 3 hours, a solution ofN-fluoro-N(phenylsulfonyl)benzenesulfonamide (13.0 g, 41.2 mmol) intetrahydrofuran (8 mL) was added drop-wise, and stirring was continuedfor 30 minutes at −78° C. The reaction mixture was then allowed toslowly warm to room temperature and stir overnight, whereupon it wasdiluted with water (20 mL) and treated with a solution of hydrogenchloride in 1,4-dioxane (4.0 M; 20.6 mL, 82.4 mmol) to hydrolyze thepresumed intermediate C18{[5-(1,3-dioxolan-2-yl)-2,6-difluorothieno[3,2-b]thiophen-3-yl](trimethyl)silane}.After the reaction mixture had stirred for 2 hours at room temperature,it was diluted with water and extracted with diethyl ether. The combinedorganic layers were dried over sodium sulfate, filtered, concentrated invacuo, and purified using silica gel chromatography (Gradient: 0% to 80%ethyl acetate in heptane) to afford C19 as a yellow oil that solidifiedover time to an orange solid. Yield: 1.48 g, 5.35 mmol, 26%. LCMS m/z277.1 [M+H]⁺. ¹H NMR (400 MHz, chloroform-d) 10.05 (s, 1H), 0.41 (d,J=0.9 Hz, 9H).

Step 2. Synthesis of 3,5-difluorothieno[3,2-b]thiophene-2-carboxylicacid (5)

Sodium dihydrogen phosphate (3.22 g, 26.8 mmol) and sodium chlorite(2.43 g, 26.9 mmol) were added to a solution of C19 (1.48 g, 5.35 mmol)in tetrahydrofuran (45 mL), water (9 mL), and 2-methyl-2-butene (9 mL),whereupon the reaction mixture was stirred at room temperature for 1hour. It was then diluted with water and extracted with ethyl acetate;the combined organic layers were dried over sodium sulfate, filtered,and concentrated in vacuo. The resulting solid {C20;3,5-difluoro-6-(trimethylsilyl)thieno[3,2-b]thiophene-2-carboxylic acid,LCMS m/z 293.0 [M+H]⁺} was dissolved in tetrahydrofuran (45 mL) andtreated sequentially with water (0.483 mL, 26.8 mmol) and a solution oftetrabutylammonium fluoride in tetrahydrofuran (1.0 M; 6.44 mL, 6.44mmol). After 5 minutes, LCMS analysis indicated that the reaction wascomplete; the reaction mixture was treated with hydrochloric acid (1 M;10.7 mL, 10.7 mmol), diluted with water, and extracted with ethylacetate. After the combined organic layers had been dried over sodiumsulfate, they were filtered, concentrated in vacuo, and subjected tosilica gel chromatography (Gradient: 0% to 20% methanol indichloromethane), followed by reversed-phase chromatography using a C18column [Gradient: 10% to 100% water (containing 0.01% trifluoroaceticacid) in acetonitrile (containing 0.01% trifluoroacetic acid)]. Uponremoval of organic solvents from the product-containing fractions invacuo, a solid precipitated. The resulting aqueous mixture was extractedwith ethyl acetate, and the combined organic layers were dried oversodium sulfate, filtered, and concentrated in vacuo. The resulting solidwas triturated with diethyl ether and heptane, then purified usingsilica gel chromatography (Gradient: 0% to 100% ethyl acetate inheptane, then 0% to 30% methanol in ethyl acetate) followed byreversed-phase chromatography on a C18 column (Gradient: 10% to 100%water in acetonitrile). The precipitated solid was collected viafiltration after removing organic solvents in vacuo from the appropriatefractions; it was then washed with water, dissolved in ethanol andfiltered. After the filtrate had been concentrated in vacuo, the residuewas triturated with ethanol and heptane to provide3,5-difluorothieno[3,2-b]thiophene-2-carboxylic acid as a white solid.Yield: 718 mg, 3.26 mmol, 61%. LCMS m/z 175.1 [(M-CO₂)—H]⁻. ¹H NMR (400MHz, DMSO-d₆) δ13.6-13.5 (br s, 1H), 7.39 (dd, J=1.7, 1.7 Hz, 1H).

TABLE 1 Method of Synthesis, Structure, Compound Name, andCharacterization Data for Examples 6-16, BT2, and BT2F. Method ofsynthesis; Non- ¹H NMR (400 MHz, DMSO- commercial d₆) δ; Mass spectrum,starting observed ion m/z [M + H]⁺ or materials HPLC retenetion time;Mass Example literature Compound spectrum m/z [M + H]⁺ Number referenceStructure Name (unless otherwise indicated) 6 Footnote 1

5-fluoro-6- methylthieno [3,2- b]thiophene- 2- carboxylic acid13.30-13.15 (br s, 1H), 8.08 (s, 1H), 2.24 (d, J = 2.0 Hz, 3H); LCMS m/z215.0 [M − H]⁻ 7 C1²

5-chloro-3- fluorothieno [3,2- b]thiophene- 2- carboxylic acid 13.7-13.6(br s, 1H), 7.70 (d, J = 1.9 Hz, 1H); LCMS m/z 191.1 (chorine isotopepattern observed [(M − CO₂) − H]⁻ 8 4³

3-chloro- 5,6- difluorothieno [3,2- b]thiophene- 2- carboxylic acid 14(br s, 1H, assumed); LCMS m/z 209.1 (chorine isotope pattern observed)[(M − CO₂) − H]⁻ 9 C2⁴

5-fluoro-3- methylthieno [3,2- b]thiophene- 2- carboxylic acid 13.2-13.1(br s, 1H), 7.33 (d, J = 1.8 Hz, 1H), 2.56 (s, 3H); LCMS m/z 215.1 [M −H]⁻ 10 Foootnote 5

6-chloro-5- fluorothieno [3,2- b]thiophene- 2- carboxylic acid 13.6-13.5(br s, 1H), 8.15 (s, 1H); LCMS m/z 235.0 (chloroine isotope patternobserved) [M − H]⁻ 11 C10⁶

5-chloro-3- (difluoromethyl) thieno [3,2- b]thiophene- 2- carboxylicacid 14.5-14.0 (br s, 1H), 7.75 (s, 1H), 7.62 (t, J_(HF) = 54.8 Hz, 1H);269.0 (chlorine isotope pattern observed) 12 C1⁷

5-chloro-6- fluorothieno [3,2- b]thiophene- 2- carboxylic acid ¹H NMR(400 MHz, methanol-d₄) δ 7.96 (d, J = 2.2 Hz, 1H); LCMS m/z 191.0(chlorine isotope pattern observed) [(M − CO₂) − H]⁻ 13 Footnote 8

5-chloro- 3,6- difluorothieno [3,2- b]thiophene- 2- carboxylic acid 2.89minutes⁹; 255.2 (chlorine isotope pattern observed) 14 Footnote 10

5-chloro-3- methylthieno [3,2- b]thiophene- 2- carboxylic acid ¹H NMR(600 MHz, methanol-d₄) δ 7.34 (s, 1H), 2.61 (s, 3H); LCMS m/z 230.9(chlorine isotope pattern observed) [M − H]⁻ 15 Footnote 11

ammonium 3-bromo-5- chlorothieno [3,2- b]thiophene- 2- carboxylate 3.08minutes⁹; 297.1 (bromo chloro isotope pattern observed) 16 Footnote 12

5-chloro-3- ethylthieno [3,2- b]thiophene- 2- carboxylic acid 7.65 (s,1H), 3.08 (q, J = 7.6 Hz, 2H), 1.21 (t, J = 7.6 Hz, 3H); LCMS m/z 245.0(chlorine isotope pattern observed) [M − H]⁻ BT2 Footnote 13

3,6- dichloro-1- benzothiophene- 2- carboxylic acid 8.33 (d, J = 1.9 Hz,1H), 7.94 (d, J = 8.7 Hz, 1H), 7.63 (dd, J = 8.7, 2.0 Hz, 1H); LCMS m/z201.2 (dichloro isotope pattern observed) [(M − CO₂) − H]⁻ BT2F Footnote13

3-chloro-6- fluoro-1- benzothiophene- 2-carboxylic acid ¹H NMR (400 MHz,methanol-d₄) δ 7.99 (dd, J = 9.0, 5.1 Hz, 1H), 7.73 (dd, J = 8.8, 2.3Hz, 1H), 7.35 (ddd, J = 9.0, 9.0, 2.4 Hz, 1H); 231.1 (chlorine isotopepattern observed) 1. Treatment of5-fluorothieno[3,2-b]thiophene-2-carboxylic acid (see Gronowitz, S.;Herslöf, M.; Senson, R.; Bondesson, G.; Magnusson, O.; Stjernström, N.E. Acta Pharm. Suec. 1978, 15, 368-381) with 2 equivalents of lithiumdiisopropylamide, followed by iodomethane, afforded Example 6. 2.Treatment of C1 with n-butyllithium and trimethylsilyl chloride provided[5-(1,3-dioxolan-2-yl)thieno[3,2-b]thiophen-2-yl](trimethyl)silane,which was reacted with n-butyllithium andN-fluoro-N-(phenylsulfonyl)benzenesulfonamide, followed bytetrabutylammonium fluoride, to afford2-(3-fluorothieno[3,2-b]thiophen-2-yl)-1,3-dioxolane. This material wasreacted with n-butyllithium and hexachloroethane to provide2-(5-chloro-3-fluorothieno[3,2-b]thiophen-2-yl)-1,3-dioxolane, which wasconverted to Example 7 using the chemistry described in Example 4 tosynthesize 4 from C16. 3. Reaction of 4 with 3 equivalents of lithiumdiisopropylamide, followed by hexachloroethane, provided Example 8. 4.Reaction of C2 with lithium diisopropylamide and iodomethane, followedby desilation with tetrabutylammonium fluoride, afforded2-(5-fluoro-3-methylthieno[3,2-b]thiophen-2-yl)-1,3-dioxolane. Thismaterial was converted to Example 9 using the method described forsynthesis of 4 from C16 in Example 4. 5. Treatment of5-fluorothieno[3,2-b]thiophene-2-carboxylic acid (see Gronowitz, S.;Herslöf, M.; Svenson, R.; Bondesson, G.; Magnusson, O.; Stjernström, N.E. Acta Pharm. Suec. 1978, 15, 368-381) with 2 equivalents of lithiumdiisopropylamide, followed by N-chlorosuccinimide, provided Example 10.6. Chlorination of C10 with N-chlorosuccinimide afforded ethyl5-chloro-3-(difluoromethyl)thieno[3,2-b]thiophene-2-carboxylate, whichwas subjected to ester hydrolysis with sodium hydroxide to provideExample 11. 7. Reaction of C1 with lithium diisopropylamide andhexachloroethane provided2-(5-chlorothieno[3,2-b]thiophen-2-yl)-1,3-dioxolane, which was thenfluorinated using n-butyllithium andN-fluoro-N-(phenylsulfonyl)benzenesulfonamide. The resulting2-(5-chloro-6-fluorothieno[3,2-b]thiophen-2-yl)-1,3-dioxolane wasconverted to Example 12 using the chemistry described in Example 4 tosynthesize 4 from C16. 8.2-(5-Chloro-3-fluorothieno[3,2-b]thiophen-2-yl)-1,3-dioxolane (seefootnote 2) was fluorinated with lithium diisopropylamide andN-fluoro-N-(phenylsulfonyl)benzenesulfonamide. The resulting2-(5-chloro-3,6-difluorothieno[3,2-b]thiophen-2-yl)-1,3-dioxollane wasconverted to Example 13 using the chemistry described in Example 4 tosynthesize 4 from C16. 9. Conditions for analytical HPLC. Column: WatersAtlantis dC18, 4.6 × 50 mm, 5 μm; Mobile phase A: 0.05% trifluoroaceticacid in water (v/v); Mobile phase B: 0.05% trifluoroacetic acid inacetonitrile (v/v); Gradient: 5.0% to 95% B, linear over 4.0 minutes;Flow rate: 2 mL/minute. 10. 3-Methylthieno[3,2-b]thiophene-2-carboxylicacid (see Deng, H.; Fang, Y.; He, M.; Hu, H.; Niu, W.; Sun, H. WO2012012278, Jan. 26, 2012) was chlorinated using N-chlorosuccinimide toafford Example 14. 11. Reaction of 3-bromothiophene-2-carbonitrile withethyl mercaptoacetate in the presence of potassium carbonate and18-crown-6 (1,4,7,10,13,16-hexaoxacyclooctadecane) provided ethyl[(2-cyanothiophen-3-yl)thio]acetate. Cyclization with lithiumbis(trimethylsilyl)amide afforded ethyl3-aminothieno[3,2-b]thiophene-2-carboxylate, which was treated with2-methyl-2-nitropropane and copper(II) bromide, and then subjected toester hydrolysis with sodium hydroxide, providing3-bromothieno[3,2-b]thiophene-2-carboxylic acid. Reaction withN-chlorosuccinimide afforded Example 15. 12. Reaction of lithiumdiisopropylamide with 2,5-dibromothiophene was followed by addition ofN-methoxy-N-methylpropanamide. The resulting1-(3,5-dibromo-2-thiophenyl)-1-propanone was converted to ethyl5-bromo-3-ethylthieno[3,2-b]thiophene-2-carboxylate with ethylmercaptoacetate in the presence of potassium carbonate and 18-crown-6.Ester hydrolysis via treatment via treatment with lithium hydroxide wasthen followed by metal-halogen exchange with n-butyllithium and reactionwith N-chlorosuccinimide to afford Example 16. 13. BT2 and BT2Fsynthesis is described in Tso, S.-C.; Gui, W.-J.; Wu, C.-Y.; Chuang, J.L.; Qi, X.; Skvorak, K. J.; Dorko, K.; Wallace, A. L.; Morlock, L. K.;Lee, B. H.; Hutson, S. M.; Strom, S. C.; Williams, N. S.; Tambar, U. K.;Wynn, R. M.; Chuang, D. T. J. Biol. Chem. 2014, 289, 20583-20593.The following protocols may of course be varied by those skilled in theart.

Protein Generation

BCKDK protein was generated using a pET vector containing from N- toC-terminus: 6×His, MBP, a TEV protease site (ENLYFQG), a biotin acceptorpeptide (GLNDIFEAQKIEWHE), and human BCKDK (residues 31-412 of theprotein pre-processing). Protein was co-expressed with GroEL-GroES inBL21(DE3) E. coli in LB media, and protein production was induced with0.5 mM IPTG and 0.5 mg/mL Larabinose at an OD₆₀₀ of 1 and grown for 16 hat 26° C. Bacteria were lysed using a Microfluidizer in 100 mM potassiumphosphate pH 7.5, 500 mM NaCl, 0.1 mM EDTA, 1% Tween-20, 0.25% TritonX-100, 10% glycerol, 1 mM DTT, and protease inhibitors. MBP-taggedprotein was purified by affinity chromatography using amylose resin, andMBP was removed from BCKDK by TEV protease incubation followed by gelfiltration chromatography in 50 mM HEPES pH 7.5, 500 mM NaCl, 300 mML-Arginine, 2 mM MgCl₂, 1 mM DTT, and 10% glycerol.

A pET vector containing E. coli LplA was expressed in BL21(DE3) E. coliin LB media, and protein production was induced with 0.75 mM IPTG at anOD₆₀₀ of 1 and grown for 16 h at 30° C. Bacteria were lysed using aMicrofluidizer in 50 mM sodium phosphate buffer pH 7.5, 350 mM NaCl, 1.5mM MgCl₂, and 1 mM DTT. LplA protein was precipitated from clarifiedlysate with 1 M ammonium sulfate and further purified by gel filtrationchromatography in 50 mM sodium phosphate pH 7.5, 350 mM NaCl, 1.5 mMMgCl₂, 1 mM DTT, and 10% glycerol.

The BCKDHE1α-E2 fusion substrate was cloned into a pET vector andcontained from N- to C-terminus: the lipoyl binding domain of E2(residues 62-160 pre-processing), a TEV protease site (LENLYFQG),residues 331-345 (pre-processing) from E1α, and 6×His (Tso, S. C. etal., J Biol Chem 2014, 289 (30), 20583-20593). The fusion substrate wasexpressed in BL21(DE3) E. coli in LB media, and protein production wasinduced with 0.75 mM IPTG at an OD₆₀₀ of 1 and grown for 16 h at 30° C.Bacteria were lysed using a Microfluidizer in 50 mM sodium phosphate pH7.5, 350 mM NaCl, 10 mM imidazole, 10% glycerol, 1 mM DTT, and proteaseinhibitors. Fusion substrate was purified by Ni-NTA affinitychromatography followed gel filtration chromatography in 50 mM sodiumphosphate pH 7.5, 350 mM NaCl, 1.5 mM MgCl₂, 1 mM DTT, and 10% glycerol.For lipoylation, fusion substrate was incubated with LplA at a 10:1(substrate:LplA) ratio in 20 mM sodium phosphate pH 7.4, 6 mM MgCl₂, 4mM ATP, 2 mM DTT, 3 mM DL-6,8-thioctic acid at 37° C. The reaction wasmonitored using an Agilent 6530 Q-TOF coupled to an Agilent 1290 UPLC.The final lipoylated fusion substrate was purified by gel filtrationchromatography in 50 mM HEPES pH 7.5, 350 mM NaCl, 1.5 mM MgCl₂, 1 mMDTT, 10% glycerol.

In Vitro FRET

BCKDK activity was monitored by phosphorylation of a HIS-tagged fusionBCKDHE1α-E2 substrate protein as described above and was detected usinga time resolved-fluorescence resonance energy transfer (TR-FRET) assaysystem. Compounds were spotted into a 384 well plate, and purified humanBCKDK protein was added to the plated compound. After incubation, theLBD-linker-E1 phosphorylation sequence was added in the presence of 15μM ATP. The reaction was terminated with EDTA. Phosphorylated substratewas recognized by the addition of rabbit anti-E1 phospho Ser293antibodies (Bethyl Laboratories—A304-672A), and the TR-FRET signal wasdeveloped by addition of anti-HIS donor molecules (Europium; PerkinElmer—AD0205, AD0110, AD0111) and anti-Rabbit acceptor molecules(Ulight; Perkin Elmer—TRF502D, TRF502M, TRF502R). Recognition ofphosphorylated E1 brought donor and acceptor molecules into closeproximity, and excitation at 320 nm caused energy transfer from theEuropium donor to the Ulight acceptor dye, which in turn generated lightat 665 nm. Signal intensity was proportional to the level ofBCKDK-mediated substrate phosphorylation. Reactions were normalized tozero percent effect with DMSO and one hundred percent effect with 600 μMRadicicol, a known BCKDK inhibitor. IC₅₀ curves were generated usingABASE software (IDBS, Boston Mass.).

Phospho BCKDHA AlphaLISA

Prior to conducting the assay, BCKDH antibodies (Bethyl A303-790A) werebiotinylated using the ChromaLink™ One-Shot Antibody Biotinylation KitB-9007-009K and phospho Ser293 BCKDHA antibodies (Bethyl A304-672A) weredirectly conjugated to AlphaLISA Acceptor Beads (custom conjugationperformed by Perkin Elmer's Lance/Delfia Custom Services, Boston Mass.).Human skeletal myocytes (Gibco A11440) were plated in a 384 well plateat a density of 7500 live cells/well and grown in skeletal muscle growthmedia containing the media supplement and chick embryo extract(Promocell C-23060 and C-23160, MP92850145). After overnight incubation,media was removed, and BCKDK inhibitors were added in assay media(growth media diluted 10-fold in PBS). After 60 minutes, the media wasremoved, the cells were washed with PBS and lysed in 10 μL of buffer(Cell Signaling #9803) containing 2 nM biotinylated total BCKDHantibodies. Samples were incubated for 60 minutes, and 5 μL of AlphaLISAacceptor beads conjugated with phospho-5293 BCKDH antibodies were added1× Alpha buffer. After a 60 minute incubation, 5 μL streptavidin donorbeads (40 μg/μL) beads were added in 1× Alpha buffer while protectingfrom light. Fluorescence was emitted when the phospho and total BCKDHantibodies were within proximity, signifying phosphorylation of S293BCKDH. Fluorescence was monitored on the Envision plate reader. The zeropercent effect was determined from DMSO treatment and the maximal effectwas assessed relative to the BCKDK inhibitor BT2. IC₅₀ curves weregenerated using ActivityBase software (IDBS, Boston Mass.).

In Table 2 assay data (IC₅₀s) are presented for the Examples below inaccordance with the above-described assays (to two (2) significantfigures as the geometric mean, based on the number of replicates tested(Number)).

TABLE 2 In Vitro FRET and Phospho BCKDHA AlphaLISA Data for Examples1-16, BT2, and BT2F. Phospho BCKDHA Example In Vitro FRET AlphaLISANumber Compound Name IC₅₀ (μM) N IC₅₀ (μM) N 13-chloro-5-fluorothieno[3,2- 0.11 18 0.72 7 b]thiophene-2-carboxylicacid 2 3-bromo-5-fluorothieno[3,2- 0.12 8 1.5 4 b]thiophene-2-carboxylicacid 3 3-(difluoromethyl)-5-fluorothieno[3,2- 0.065 8 1.8 4b]thiophene-2-carboxylic acid 4 5,6-difluorothieno[3,2- 0.058 12 0.39 7b]thiophene-2-carboxylic acid 5 3,5-difluorothieno[3,2- 0.14 11 0.41 5b]thiophene-2-carboxylic acid 6 5-fluoro-6-methylthieno[3,2- 0.23 3 4.23 b]thiophene-2-carboxylic acid 7 5-chloro-3-fluorothieno[3,2- 0.26 31.1 3 b]thiophene-2-carboxylic acid 8 3-chloro-5,6-difluorothieno[3,2-0.26 4 1.8 4 b]thiophene-2-carboxylic acid 95-fluoro-3-methylthieno[3,2- 0.28 3 1.6 3 b]thiophene-2-carboxylic acid10 6-chloro-5-fluorothieno[3,2- 0.33 3 3.4 3 b]thiophene-2-carboxylicacid 11 5-chloro-3-(difluoromethyl)thieno[3,2- 0.37 3 2.2 3b]thiophene-2-carboxylic acid 12 5-chloro-6-fluorothieno[3,2- 0.40 4 1.22 b]thiophene-2-carboxylic acid 13 5-chloro-3,6-difluorothieno[3,2- 0.473 1.9 2 b]thiophene-2-carboxylic acid 14 5-chloro-3-methylthieno[3,2-0.80 3 Not Tested b]thiophene-2-carboxylic acid 15 ammonium3-bromo-5-chlorothieno[3,2- 0.84 3 2.8 3 b]thiophene-2-carboxylate 165-chloro-3-ethylthieno[3,2- 0.88 3 3.6 3 b]thiophene-2-carboxylic acidBT2 3,6-dichloro-1-benzothiophene-2- 1.3 115 4.5 19 carboxylic acid BT2F3-chloro-6-fluoro-1-benzothiophene- 0.81 3 Not Tested 2-carboxylic acid

Diabetic Animal Model

Mice fed 60% high fat diet (Research Diets 12492) were dosed PO withExample 1 for one day, fasted overnight, and blood glucose was measuredwith an alpha track glucometer. The animals were dosed again PO withExample 1 the next morning, and one hour later, blood glucose wasmeasured again immediately using an alpha track glucometer (Zoetis,Parsippany, N.J.) to assess fasting glucose levels prior to oral gavageof 1 g/kg dextrose. Blood glucose was measured 15, 30, 60, and 120minutes after the gavage, and the data were plotted and analyzed as areaunder the curve using GraphPad Prism 8.0 (GraphPad Software, La Jolla,Calif.). Blood was concomitantly collected in EDTA tubes at the 0, 15and 30 minute time points, spun down at 10K RPM for 10 minutes. Foranimals that were dosed with vehicle or Example 1 as above, mean±SEMfasting plasma glucose levels were 237±5 (vehicle, n=18), 230±8 (20mg/kg, n=18), 221±4 (60 mg/kg, n=40), 206±5 mg/dL (180 mg/kg, n=19).Area under the curve for the glucose tolerance test as percent ofvehicle treated group was 100.0±3 (vehicle, n=29), 100±4 (20 mg/kg,n=18), 87±2 (60 mg/kg, n=30), 78±2 (180 mg/kg, n=16).

Heart Failure Rat Model

Dahl salt sensitive male rats (Charles River strain SS/JrHsdMcwiCrl)were fed control diet or 6% high salt diet (iD03121701-AIN-76a rodentdiet with added 6% NaCl) for 21 weeks in total. At week 5, the high saltdiet-fed rats were dosed PO with 100 mg/kg BT2 or vehicle once daily forthe last 16 weeks of study. Echocardiography was performed at week 18(myocardial performance index (MPI): control diet 0.567±0.034, highsalt+vehicle 0.810±0.039, high salt+BT2 0.660±0.030; Isovolumicrelaxation time (IVRT): control diet 23.154±0.60 ms, high salt+vehicle36.507±2.20 ms, high salt+BT2 31.605±1.78 ms; Intraventricular septalthickness at diastole (IVDd): control 2.03±0.088 mm, high salt+vehicle2.877±0.110 mm, high salt+BT2 2.489±0.089 mm). NT-pro-BNP (MSD K153JKD,control 294.9±26.04 μg/mL, high salt+vehicle 1003.0±200.8 μg/mL, highsalt+BT2 503.4±84.96 μg/mL), and proANP (MSD K153MBD; control 33.50±5.4ng/mL, high salt 65.19±8.3 ng/mL, high salt+BT2 38.81±7.0 ng/mL) levelswere measured in plasma using MSD assays at the terminal time point.Heart weights were measured at euthanasia and normalized to tibia length(heart/tibia control 0.033±0.001 g/mm; high salt+vehicle 0.042±0.001g/mm, high salt+BT2 0.038±0.001 g/mm).

Heart Failure Mouse Model

Male adult mice (8-16-week-old, Charles River strain C57BL6/NCrI) wereused for transverse aortic constriction. One week prior to surgery,animals were dosed with BT2 (40 mg/kg) or vehicle. On the day ofsurgery, animals were anesthetized, the chest cavity was opened, theaortic area was cleaned, and a silk suture was placed around thetransverse aorta. Sham mice were not tied, and TAC mice had the suturetied around a needle. Mice were allowed to recover and were dosed eitherorally with BT2 (40 mg/kg) once daily or vehicle. Echocardiography wasperformed serially. Heart weights and lung weights were measured ateuthanasia. Data obtained with BT2 have been reported in Sun et al,Circulation. 2016 May 24; 133(21):2038-49. doi:10.1161/CIRCULATIONAHA.115.020226.

Powder X-Ray Diffraction

Powder X-ray diffraction analysis for the compound of Example 1 wasconducted using a Bruker AXS D4 Endeavor diffractometer equipped with aCu radiation source. The divergence slit was set at 0.6 mm while thesecondary optics used variable slits. Diffracted radiation was detectedby a PSD-Lynx Eye detector. The X-ray tube voltage and amperage were setto 40 kV and 40 mA respectively. Data was collected in the Theta-2Thetagoniometer at the Cu wavelength from 3.0 to 40.0 degrees 2-Theta using astep size of 0.020 degrees and a step time of 0.3 second. Samples wereprepared by placing them in a silicon low background sample holder androtated during collection.

For the powder X-ray diffraction analysis of the compound of Example 2,a Bruker AXS D8 Endeavor diffractometer equipped with a Cu radiationsource was used. The divergence slit was set at 3 mm continuousillumination. Diffracted radiation was detected by a LYNXEYE EX detectorwith motorized slits. Both primary and secondary equipped with 2.5soller slits. The X-ray tube voltage and amperage were set at 40 kV and40 mA respectively. Data was collected in the Theta-Theta goniometer ina locked couple scan at Cu K-alpha (average) wavelength from 3.0 to 40.0degrees 2-Theta with an increment of 0.02 degrees, using a scan speed of0.5 seconds per step. Samples were prepared by placement in a siliconlow background sample holder.

Data were collected from both instruments for Examples 1 and 2 usingBruker DIFFRAC Plus software and analysis was performed by EVA diffractplus software. The PXRD data file was not processed prior to peaksearching. Using the peak search algorithm in the EVA software, peaksselected with a threshold value of 1 were used to make preliminary peakassignments. To ensure validity, adjustments were manually made; theoutput of automated assignments was visually checked and peak positionswere adjusted to the peak maximum. Peaks with relative intensity of 3%were generally chosen. The peaks which were not resolved or wereconsistent with noise were not selected. A typical error associated withthe peak position from PXRD stated in USP up to +1-0.2° 2-Theta(USP-941). FIGS. 1 and 2 show the characteristic x-ray powderdiffraction patterns of crystalline form 1 of Example 1 and crystallineform 1 of Example 2, respectively. The PXRD data from these figures arefurther described below.

TABLE 3a Key PXRD peaks to characterize crystalline material of Example1, Form 1 and Example 2, Form 1 Example 1, Form 1 Example 2, Form 1Angle 2Θ (°) Angle 2Θ (°) 11.8, 14.4, 15.5, 18.8 6.4, 14.3, 15.4, 19.0

TABLE 3b PXRD peaks for crystalline material of Example 1, Form 1 Angle2Θ (°) Relative intensity (%) 11.8 17.0 12.5 48.7 14.4 3.5 15.5 100.018.8 38.2 23.3 10.5 24.1 3.6 24.6 3.6 25.2 87.0 25.8 26.6 26.4 8.3 26.66.8 27.4 4.1 29.9 5.3 31.3 5.6 31.7 6.4 33.2 20.3 35.2 15.8 35.7 4.936.0 7.0 37.3 8.7 38.2 5.1

TABLE 3c PXRD peaks for crystalline material of Example 2, Form 1 Angle2Θ (°) Relative intensity (%) 6.4 72.4 7.6 4.4 7.3 6.8 12.7 67.2 14.313.9 15.4 6.5 19.0 100.0 23.0 10.7 23.4 4.9 24.2 3.4 25.4 69.3 25.8 28.626.1 16.7 27.1 6.6 27.4 5.3 30.0 10.6 31.6 10.6 33.1 17.4 34.6 4.5 35.15.8 36.1 9.5 36.7 3.8

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application for all purposes.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

1. A compound of Formula I

wherein R¹ is H, bromo, chloro, fluoro, (C₁-C₂)alkyl, or(C₁-C₂)fluoroalkyl, R² is fluoro or chloro, wherein if R¹ is chloro andR³ is H then R² is fluoro; and R³ is H, chloro, fluoro, methyl, or(C₁)fluoroalkyl, wherein if R¹ is H then R³ is chloro, fluoro, methyl or(C₁)fluoroalkyl, or a pharmaceutically acceptable salt of said compound2. A compound of claim 1 wherein R¹ is H, bromo, chloro, ordifluoro(C₁)alkyl; R² is fluoro; and R³ is H or fluoro wherein if R¹ isH then R³ is fluoro; or a pharmaceutically acceptable salt of saidcompound.
 3. A compound of claim 1 selected from the group consistingof: 3-chloro-5-fluorothieno[3,2-b]thiophene-2-carboxylic acid;3-bromo-5-fluorothieno[3,2-b]thiophene-2-carboxylic acid;3-(difluoromethyl)-5-fluorothieno[3,2-b]thiophene-2-carboxylic acid;5,6-difluorothieno[3,2-b]thiophene-2-carboxylic acid; and3,5-difluorothieno[3,2-b]thiophene-2-carboxylic acid; or apharmaceutically acceptable salt thereof.
 4. A compound wherein thecompound is

or a pharmaceutically acceptable salt of said compound.
 5. A compoundwherein the compound is3-chloro-5-fluorothieno[3,2-b]thiophene-2-carboxylic acid or apharmaceutically acceptable salt thereof.
 6. A compound wherein thecompound is 3-bromo-5-fluorothieno[3,2-b]thiophene-2-carboxylic acid ora pharmaceutically acceptable salt thereof.
 7. A method of treatingfatty liver, nonalcoholic fatty liver disease, nonalcoholicsteatohepatitis, nonalcoholic steatohepatitis with liver fibrosis,nonalcoholic steatohepotitis with cirrhosis or nonalcoholicsteatohepatitis with cirrhosis and hepatocellular carcinoma comprisingadministering to a human in need of such treatment a therapeuticallyeffective amount of a compound of claim 4 or a pharmaceuticallyacceptable salt thereof.
 8. The method as recited in claim 7 whereinnonalcoholic steatohepatitis is treated.
 9. A method of treating heartfailure, congestive heart failure, coronary heart disease, peripheralvascular disease, renovascular disease, pulmonary hypertension,vasculitis, acute coronary syndromes and modification of cardiovascularrisk comprising administering to a human in need of such treatment atherapeutically effective amount of a compound of claim 4 or apharmaceutically acceptable salt thereof.
 10. The method as recited inclaim 9 wherein heart failure is treated.
 11. A method of treating TypeI diabetes, Type II diabetes mellitus, idiopathic Type I diabetes (TypeIb), latent autoimmune diabetes in adults (LADA), early-onset Type 2diabetes (EOD), youth-onset atypical diabetes (YOAD), maturity onsetdiabetes of the young (MODY), malnutrition-related diabetes, gestationaldiabetes, coronary heart disease, ischemic stroke, restenosis afterangioplasty, peripheral vascular disease, intermittent claudication,myocardial infarction, dyslipidemia, post-prandial lipemia, conditionsof impaired glucose tolerance (IGT), conditions of impaired fastingplasma glucose, metabolic acidosis, ketosis, arthritis, diabeticretinopathy, macular degeneration, cataract, diabetic nephropathy,glomerulosclerosis, chronic renal failure, diabetic neuropathy,metabolic syndrome, syndrome X, hyperglycemia, hyperinsulinemia,hypertriglyceridemia, insulin resistance, impaired glucose metabolism,skin and connective tissue disorders, foot ulcerations and ulcerativecolitis, endothelial dysfunction and impaired vascular compliance, hyperapo B lipoproteinemia, and maple syrup urine disease comprisingadministering to a human in need of such treatment a therapeuticallyeffective amount of a compound of claim 4 or a pharmaceuticallyacceptable salt thereof.
 12. The method as recited in claim 11 whereinType II diabetes mellitus is treated.
 13. A method of treatinghepatocellular carcinoma, kidney renal clear cell carcinoma, head andneck squamous cell carcinoma, colorectal adenocarcinoma, mesothelioma,stomach adenocarcinoma, adrenocortical carcinoma, kidney papillary cellcarcinoma, cervical and endocervical carcinoma, bladder urothelialcarcinoma, lung adenocarcinoma comprising administering to a human inneed of such treatment a therapeutically effective amount of a compoundof claim 4 or a pharmaceutically acceptable salt thereof.
 14. The methodas recited in claim 13 wherein hepatocellular carcinoma or colorectaladenocarcinoma is treated.
 15. A pharmaceutical composition whichcomprises a therapeutically effective amount of a compound of claim 4 ora pharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier, vehicle or diluent.
 16. A pharmaceutical combinationcomposition comprising: a therapeutically effective amount of acomposition comprising: a first compound, said first compound being acompound of any of claim 4, or a pharmaceutically acceptable saltthereof; a second compound, said second compound being an anti-diabeticagent; a non-alcoholic steatohepatitis treatment agent, a non-alcoholicfatty liver disease treatment agent or an anti-heart failure treatmentagent and a pharmaceutical carrier, vehicle or diluents.
 17. Thepharmaceutical combination composition as recited in claim 16 whereinsaid second compound is4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4′-piperidine]-t-carbonyl)-6-methoxypyridin-2-yl)benzoicacid;[(1R,5S,6R)-3-{2-[(2S)-2-methylazetidin-1-yl]-6-(trifluoromethyl)pyrimidin-4-yl}-3-azabicyclo[3.1.0]hex-6-yl]aceticacid;2-[(1R,3R,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylicacid;(S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide,or2-[(4-{6-[(4-cyano-2-fluorobenzyl)oxy]pyridin-2-yl}piperidin-1-yl)methyl]-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylicacid, or a pharmaceutically acceptable salt thereof.
 18. Thepharmaceutical combination composition as recited in claim 16 whereinsaid non-alcoholic steatohepatitis treatment agent or non-alcoholicfatty liver disease treatment agent is an ACC inhibitor, a KHKinhibitor, a DGAT-2 inhibitor, an FXR agonist, metformin, incretinanalogs, or an incretin receptor modulator.
 19. The pharmaceuticalcombination composition as recited in claim 16 wherein saidanti-diabetic agent is an SGLT-2 inhibitor, metformin, incretin analogs,an incretin receptor modulator, a DPP-4 inhibitor, or a PPAR agonist.20. The pharmaceutical combination composition as recited in claim 19wherein said anti-diabetic agent is metfomin, sitagliptin orertuglifozin.
 21. The pharmaceutical combination composition as recitedin claim 16 wherein said anti-heart failure agent is an ACE inhibitor,an SGLT2 inhibitor, an angiotensin receptor blocker, a neprilysininhibitor, an angiotensin-receptor blocker/neprilysin inhibitor, a betaadrenergic receptor blocker, a calcium channel blocker, or avasodilator.
 22. A crystal comprising a compound of claim 4 having thestructure:

or a pharmaceutically acceptable salt thereof.
 23. The crystal of claim22 having a powder x-ray diffraction pattern comprising 2-theta valuesof (CuKα radiation, wavelength of 1.54056 Å) 11.8±0.2, 14.4±0.2,15.5±0.2, and 18.8±0.2.
 24. A crystal comprising a compound of claim 4having the structure:

or a pharmaceutically acceptable salt thereof.
 25. The crystal of claim24 having a powder x-ray diffraction pattern comprising 2-theta valuesof (CuKα radiation, wavelength of 1.54056 Å) 6.4±0.2, 14.3±0.2,15.4±0.2, and 19.0±0.2.